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Although the field of fungal infections advanced tremendously, diagnosis of invasive pulmonary aspergillosis (IPA) in immunocompromised patients continues to be a challenge. Since IPA is a multifactorial disease, investigation from different aspects may provide new insights, helpful for improving IPA diagnosis. This work aimed to characterize the human immune response to Aspergillus fumigatus in a multilevel manner to identify characteristic molecular candidates and risk factors indicating IPA, which may in the future support already established diagnostic assays. We combined in vitro studies using myeloid cells infected with A. fumigatus and longitudinal case-control studies investigating patients post allogeneic stem cell transplantation (alloSCT) suffering from IPA and their match controls.
Characteristic miRNA and mRNA signatures indicating A. fumigatus-infected monocyte-derived dendritic cells (moDCs) demonstrated the potential to differentiate between A. fumigatus and Escherichia coli infection. Transcriptome and protein profiling of alloSCT patients suffering from IPA and their matched controls revealed a distinctive IPA signature consisting of MMP1 induction and LGAL2 repression in combination with elevated IL-8 and caspase-3 levels. Both, in vitro and case-control studies, suggested cytokines, matrix-metallopeptidases and galectins are important in the immune response to A. fumigatus. Identified IPA characteristic molecular candidates are involved in numerous processes, thus a combination of these in a distinctive signature may increase the specificity. Finally, low monocyte counts, severe GvHD of the gut (grade ≥ 2) and etanercept administration were significantly associated with IPA diagnosis post alloSCT. Etanercept in monocyte-derived macrophages (MDM) infected with A. fumigatus downregulates genes involved in the NF-κB and TNF-α pathway and affects the secretion of CXCL10.
Taken together, identified characteristic molecular signatures and risk factors indicating IPA may in the future in combination with established fungal biomarkers overcome current diagnostic challenges and help to establish tailored antifungal therapy. Therefore, further multicentre studies are encouraged to evaluate reported findings.
Multiple myeloma is a bone marrow plasma cell tumor which is supported by the external growth factors APRIL and IL-6, among others. Recently, we identified eosinophils and megakaryocytes to be functional components of the micro-environmental niches of benign bone marrow plasma cells and to be important local sources of these cytokines. Here, we investigated whether eosinophils and megakaryocytes also support the growth of tumor plasma cells in the MOPC315. BM model for multiple myeloma. As it was shown for benign plasma cells and multiple myeloma cells, IL-6 and APRIL also supported MOPC315. BM cell growth in vitro, IL-5 had no effect. Depletion of eosinophils in vivo by IL-5 blockade led to a reduction of the early myeloma load. Consistent with this, myeloma growth in early stages was retarded in eosinophil-deficient Delta dblGATA-1 mice. Late myeloma stages were unaffected, possibly due to megakaryocytes compensating for the loss of eosinophils, since megakaryocytes were found to be in contact with myeloma cells in vivo and supported myeloma growth in vitro. We conclude that eosinophils and megakaryocytes in the niches for benign bone marrow plasma cells support the growth of malignant plasma cells. Further investigations are required to test whether perturbation of these niches represents a potential strategy for the treatment of multiple myeloma.
Dendritische Zellen stellen ein essentielles Bindeglied zwischen angeborener und adaptiver Immunität dar. Sie stammen aus dem Knochenmark und bilden ein Netzwerk von heterogenen Zellpopulationen. In peripheren Geweben liegen sie als unreife Zellen mit einem hohen Potential zur Aufnahme und Prozessierung von Mikroorganismen vor. Nach der Aufnahme von eindringenden Mikroorganismen beginnen dendritische Zellen jedoch, sich von einem prozessierenden in ein präsentierendes Stadium zu differenzieren, und wandern zu den sekundären lymphatischen Organen, um dort den naïven T-Zellen die mikrobiellen Antigene zu präsentieren. Die gerichtete Wanderung von dendritischen Zellen ist hierbei ein zentraler Bestandteil der immunstimulatorischen und -modulatorischen Funktion dieser Zellen. Eine essentielle Rolle bei diesem Migrationsverhalten spielen chemotaktische Zytokine (Chemokine). Chemokine sind Proteine mit einem niedermolekularen Gewicht (8-10 kDa), welche anhand ihres strukturellen Cysteinmotifs in vier Gruppen unterteilt und entweder als CXC-, CC-, C- und CX3C- Chemokine oder als α-, β-, δ- und γ-Chemokine bezeichnet werden. Arbeiten der eigenen Arbeitsgruppe am Modell der kutanen Leishmaniose haben jedoch gezeigt, dass die Funktion von Chemokinen weitaus mehr umfasst, als lediglich die zielgerichtete Steuerung der Migration immunologisch wichtiger Zellen. So konnte in diesen Studien nicht nur gezeigt werden, dass das Muster der Expression von Chemokinen mit der Schwere des Krankheitsbildes korreliert, sondern auch, dass das Chemokin CCL2/MCP-1 in der Lage ist, einen direkten Einfluss auf die intrazelluläre Erregerabwehr zu nehmen. Diese Arbeiten bezogen sich jedoch auf humane Hautbiopsien und aus Humanblut isolierten Zellen. Eine detaillierte Analyse des Infektionsverlaufes unter definierten Bedingungen (konstante Infektionsdosis und -art, kontrollierte Infektionsdauer, Verwendung von klonierten Parasiten, einheitlicher genetischer Hintergrund des Wirts) ist bei Patienten jedoch nicht möglich. Deshalb wurde die experimentelle Leishmanieninfektion von Inzuchtmäusen (suszeptible BALB/c- und resistente C57BL/6-Mäuse) herangezogen, um die Kinetik der Chemokinexpression und deren Korrelation mit dem Verlauf der kutanen Leishmaniose zu bestimmen. Die Arbeiten dieser Doktorarbeit zeigen erstmals, dass ebenso wie im humanen System bei der experimentellen Leishmaniose in der Maus die Fähigkeit zur Abwehr dieses Erregers mit einer verstärkten Expression von CCL2/MCP-1 in der infizierten Haut korreliert. Des Weiteren konnte zwar eine CCL2/MCP-1-induzierte leishmanizide Wirkung in murinen Makrophagen festgestellt werden, ein vergleichbarer Effekt blieb bei Langerhans-Zellen, den dendritischen Zellen der Haut, jedoch aus. Weiterhin sollte der Einfluss einer Leishmanieninfektion auf das CCL2/MCP-1- induzierte Wanderungsverhalten von Langerhans-Zellen untersucht werden, da aus der Literatur bekannt ist, dass das Chemokin CCL2/MCP-1 die Migration dendritischer Zellen induziert. Die Studien der Doktorarbeit ergaben hierbei, dass eine Infektion mit Leishmania major zu einer signifikanten Verminderung der durch CL2/MCP-1 oder CCL3/MIP-1α induzierten Migration von Langerhans-Zellen führt. Eine mögliche Ursache für dieses Resultat war hierbei in dem Einfluss einer Infektion mit Leishmanien auf die Expression von Chemokinrezeptoren in dendritischen Zellen zu finden. Anschließende Untersuchungen der mRNA-Expression dieser Rezeptoren konnten diese Vermutung bestätigen. So wurde nach einer Infektion von dendritischen Zellen mit L. major eine Reduktion der mRNA-Expression der Chemokinrezeptoren CCR2 und CCR5 festgestellt. Anschließende FACS-Analysen und Studien mit Hilfe des konfokalen Lasermikroskops führten zu einem ähnlichen Resultat. Interessanterweise bewirkt die Infektion mit L. major andererseits eine Hochregulation der mRNA-Expression von CCR7 in dendritischen Zellen. Von diesem Rezeptor ist bekannt, dass er die Chemokine CCL19/MIP-3β und CCL21/6Ckine, welche im Lymphknoten konstitutiv exprimiert werden, mit großer Affinität bindet und für die zielgerichtete Migration dendritischer Zellen in dieses Organ essentiell ist. Weitere Versuche ergaben zudem eine verstärkte CXCL10/IP-10-mRNA-Expression in dendritischen Zellen aus L. major-resistenten C57BL/6-Mäusen, welche bei dendritischen Zellen aus BALB/c-Mäusen nicht festgestellt worden ist. Zusammenfassend konnte in dieser Doktorarbeit gezeigt werden, dass eine Infektion dendritischer Zellen mit L. major sowohl bei suszeptiblen als auch bei resistenten Mäusen zu einer Modulation der Expression von Chemokinrezeptoren führt, die sowohl für die Lokalisation der Zellen in den entzündeten Hautarealen verantwortlich sind (CCR2 und CCR5), als auch ihre Wanderung in die Lymphknoten steuern CCR7). Darüber hinaus lässt die wirtsspezifische Modulation des Chemokins CXCL10/IP-10 in resistenten Tieren vermuten, dass sie zur Kontrolle der Infektion beiträgt.
Abstract
In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-γ or IFN-γ and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection.
Author Summary
The clinical symptoms caused by infections with Leishmania parasites range from self-healing cutaneous to uncontrolled visceral disease and depend not only on the parasite species but also on the type of the host's immune response. It is estimated that 350 million people worldwide are at risk, with a global incidence of 1–1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. Murine leishmaniasis is the best-characterized model to elucidate the mechanisms underlying resistance or susceptibility to Leishmania major parasites in vivo. Using T cell-specific and macrophage-specific mutant mice, we demonstrate that abrogating the secretion of the immunosuppressive cytokine IL-10 by T cells is sufficient to render otherwise susceptible mice resistant to an infection with the pathogen. The healing phenotype is accompanied by an elevated specific inflammatory immune response very early after infection. We further show that dendritic cell-based vaccination against leishmaniasis suppresses the early secretion of IL-10 following challenge infection. Thus, our study unravels a molecular mechanism critical for host immune defense, aiding in the development of an effective vaccine against leishmaniasis.
Parent-of-origin imprints have been implicated in the regulation of neural differentiation and brain development. Previously we have shown that, despite the lack of a paternal genome, human parthenogenetic (PG) embryonic stem cells (hESCs) can form proliferating neural stem cells (NSCs) that are capable of differentiation into physiologically functional neurons while maintaining allele-specific expression of imprinted genes. Since biparental ("normal") hESC-derived NSCs (N NSCs) are targeted by immune cells, we characterized the immunogenicity of PG NSCs. Flow cytometry and immunocytochemistry revealed that both N NSCs and PG NSCs exhibited surface expression of human leukocyte antigen (HLA) class I but not HLA-DR molecules. Functional analyses using an in vitro mixed lymphocyte reaction assay resulted in less proliferation of peripheral blood mononuclear cells (PBMC) with PG compared with N NSCs. In addition, natural killer (NK) cells cytolyzed PG less than N NSCs. At a molecular level, expression analyses of immune regulatory factors revealed higher HLA-G levels in PG compared with N NSCs. In line with this finding, MIR152, which represses HLA-G expression, is less transcribed in PG compared with N cells. Blockage of HLA-G receptors ILT2 and KIR2DL4 on natural killer cell leukemia (NKL) cells increased cytolysis of PG NSCs. Together this indicates that PG NSCs have unique immunological properties due to elevated HLA-G expression.
Background
Adrenocortical tumors comprise frequent adenomas (ACA) and rare carcinomas (ACC). Human cytochrome P450 2W1 (CYP2W1) is highly expressed in some cancers holding the potential to activate certain drugs into tumor cytotoxins.
Objective
To investigate the CYP2W1 expression in adrenal samples and its relationship with clinical outcome in ACC.
Material and Methods
CYP2W1 expression was investigated by qRT-PCR in 13 normal adrenal glands, 32 ACA, 25 ACC, and 9 different non-adrenal normal tissue samples and by immunohistochemistry in 352 specimens (23 normal adrenal glands, 33 ACA, 239 ACC, 67 non-adrenal normal or neoplastic samples).
Results
CYP2W1 mRNA expression was absent/low in normal non-adrenal tissues, but high in normal and neoplastic adrenal glands (all P<0.01 vs non-adrenal normal tissues). Accordingly, CYP2W1 immunoreactivity was absent/low (H-score 0–1) in 72% of non-adrenal normal tissues, but high (H-score 2–3) in 44% of non-adrenal cancers, in 65% of normal adrenal glands, in 62% of ACAs and in 50% of ACCs (all P<0.001 vs non-adrenal normal tissues), being significantly increased in steroid-secreting compared to non-secreting tumors. In ACC patients treated with mitotane only, high CYP2W1 immunoreactivity adjusted for ENSAT stage was associated with longer overall survival and time to progression (P<0.05 and P<0.01, respectively), and with a better response to therapy both as palliative (response/stable disease in 42% vs 6%, P<0.01) or adjuvant option (absence of disease recurrence in 69% vs 45%, P<0.01).
Conclusion
CYP2W1 is highly expressed in both normal and neoplastic adrenal glands making it a promising tool for targeted therapy in ACC. Furthermore, CYP2W1 may represent a new predictive marker for the response to mitotane treatment.
Objectives
The severity of Coronavirus Disease 2019 (COVID-19) is largely determined by the immune response. First studies indicate altered lymphocyte counts and function. However, interactions of pro- and anti-inflammatory mechanisms remain elusive. In the current study we characterized the immune responses in patients suffering from severe COVID-19-induced acute respiratory distress syndrome (ARDS).
Methods
This was a single-center retrospective study in patients admitted to the intensive care unit (ICU) with confirmed COVID-19 between March 14th and May 28th 2020 (n = 39). Longitudinal data were collected within routine clinical care, including flow-cytometry of lymphocyte subsets, cytokine analysis and growth differentiation factor 15 (GDF-15). Antibody responses against the receptor binding domain (RBD) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike protein were analyzed.
Results
All patients suffered from severe ARDS, 30.8% died. Interleukin (IL)-6 was massively elevated at every time-point. The anti-inflammatory cytokine IL-10 was concomitantly upregulated with IL-6. The cellular response was characterized by lymphocytopenia with low counts of CD8+ T cells, natural killer (NK) and naïve T helper cells. CD8+ T and NK cells recovered after 8 to 14 days. The B cell system was largely unimpeded. This coincided with a slight increase in anti-SARS-CoV-2-Spike-RBD immunoglobulin (Ig) G and a decrease in anti-SARS-CoV-2-Spike-RBD IgM. GDF-15 levels were elevated throughout ICU treatment.
Conclusions
Massively elevated levels of IL-6 and a delayed cytotoxic immune defense characterized severe COVID-19-induced ARDS. The B cell response and antibody production were largely unimpeded. No obvious imbalance of pro- and anti-inflammatory mechanisms was observed, with elevated GDF-15 levels suggesting increased tissue resilience.
The initial stages of the interaction between the host and Aspergillus fumigatus at the alveolar surface of the human lung are critical in the establishment of aspergillosis. Using an in vitro bilayer model of the alveolus, including both the epithelium (human lung adenocarcinoma epithelial cell line, A549) and endothelium (human pulmonary artery epithelial cells, HPAEC) on transwell membranes, it was possible to closely replicate the in vivo conditions. Two distinct sub-groups of dendritic cells (DC), monocyte-derived DC (moDC) and myeloid DC (mDC), were included in the model to examine immune responses to fungal infection at the alveolar surface. RNA in high quantity and quality was extracted from the cell layers on the transwell membrane to allow gene expression analysis using tailored custom-made microarrays, containing probes for 117 immune-relevant genes. This microarray data indicated minimal induction of immune gene expression in A549 alveolar epithelial cells in response to germ tubes of A. fumigatus. In contrast, the addition of DC to the system greatly increased the number of differentially expressed immune genes. moDC exhibited increased expression of genes including CLEC7A, CD209 and CCL18 in the absence of A. fumigatus compared to mDC. In the presence of A. fumigatus, both DC subgroups exhibited up-regulation of genes identified in previous studies as being associated with the exposure of DC to A. fumigatus and exhibiting chemotactic properties for neutrophils, including CXCL2, CXCL5, CCL20, and IL1B. This model closely approximated the human alveolus allowing for an analysis of the host pathogen interface that complements existing animal models of IA.
Inflammation and dysregulation of the immune system are hallmarks of several neurodegenerative diseases. An activated immune response is considered to be the cause of myelin breakdown in demyelinating disorders. In the peripheral nervous system (PNS), myelin can be degraded in an autophagy-dependent manner directly by Schwann cells or by macrophages, which are modulated by T-lymphocytes. Here, we show that the NF-κB activator Pleckstrin homology containing family member 5 (Plekhg5) is involved in the regulation of both Schwann cell autophagy and recruitment of T-lymphocytes in peripheral nerves during motoneuron disease. Plekhg5-deficient mice show defective axon/Schwann cell units characterized by myelin infoldings in peripheral nerves. Even at late stages, Plekhg5-deficient mice do not show any signs of demyelination and inflammation. Using RNAseq, we identified a transcriptional signature for an impaired immune response in sciatic nerves, which manifested in a reduced number of CD4\(^+\) and CD8\(^+\) T-cells. These findings identify Plekhg5 as a promising target to impede myelin breakdown in demyelinating PNS disorders.
Rolle der gammadelta T-Zellen in der Immunantwort bei Patienten mit gastrointestinalen Tumoren
(2022)
Zusammenfassend konnte im Rahmen der vorliegenden Arbeit die Frage nach der Fähigkeit der selektiven Stimulierung mittels des Phosphorantigens HMBPP und den beiden BTN3 Antikörpern bestätigt werden. Es konnte zudem wie erwartet hierbei ein Unterschied zwischen den beiden Kohorten detektiert werden. Dabei zeigte die Kohorte der Normalspender erwartungsgemäß eine stärkere Aktivierungs- sowie Proliferations-fähigkeit. Normalspender ließen sich signifikant besser mit HMBPP aktivieren und bei bestimmter Konzentration signifikant besser proliferieren, bei BTN3A und sc20.1 konnten keine signifikanten Unterschiede ermittelt werden, allerdings anhand der Mittelwerte eine deutlich stärkere Aktivierung und Proliferation aufgezeigt werden. Außerdem konnten interessante interindividuelle Unterschiede detektiert werden, die neue Erkenntnisse brachten. Mit Hilfe der untersuchten Oberflächenmoleküle CD45RA und CD27 und der Einteilung der gammadelta T-Zellen in unterschiedliche Subgruppen konnten so mögliche Erklärungen für die Unterschiede zwischen den Kohorten aufgezeigt werden. Normalspender zeigten signifikant höhere Anteile an naiven gammadelta T-Zellen und nicht signifikant höhere Anteile an central memory T-Zellen, demnach eine deutliche Verschiebung in Richtung nicht differenzierter Subsets, wohingegen die Tumorkohorte signifikant höhere effector memory T-Zellen aufwiesen und somit eine deutliche Verschiebung in Richtung differenzierter Subsets. Dadurch kann erklärt werden, weshalb Normalspender besser aktiviert werden und besser proliferieren können. Auch die Einteilung in unterschiedliche Profile 1-6 anhand CD28, CD27 und CD16 lieferte Gründe für den Unterschied zwischen den Kohorten, wobei Normalspender der Gruppe 1 und 2, Tumorpatienten der Gruppe 3 und 4 angehörten. Durch Ermittlung weiterer signifikanter Änderungen einiger exprimierter Oberflächenmoleküle CD39, CD161 und PD1 wurde mit Hilfe der vorliegenden Arbeit bekräftigt, dass einige Faktoren betrachtet werden müssen, die die Proliferation und Aktivierung der gammadelta T-Zellen positiv und negativ beeinflussen können. Es konnte jedoch auch erneut verdeutlicht werden, wie komplex und weitgreifend der Aktivierungsmechanismus, die damit verbundene Expansion und die Auslösung der einzelnen Effektorfunktionen ist.