Wirkung von Interleukin-3 auf humane B-Zellen und Regulation ihrer Interleukin-3 Rezeptor Expression

Die Zielsetzung dieser Arbeit war die Untersuchung der Auswirkung von Interleukin-3 auf humane B-Lymphozyten und die Regulation des Zytokinrezeptors auf den Zielzellen. Spezielles Augenmerk wurde dabei auf die Proliferation, Ausdifferenzierung und Interleukinproduktion der B-Zellen gelegt. Wir bestimmten den optimalen Auswertungszeitpunkt der B-Zell-Proliferation nach sechstägiger Inkubation der B-Zellen mit verschiedenen Stimulanzien. Als besonders effektive Stimulanzien der B-Zell-Proliferation konnten wir CPG, CD40L + aHA und anti-CD180 identifizieren. Die Kombination mit IL-3 konnte sowohl bei diesen Stimulanzien, als auch bei den verwendeten Substanzen CSC, F(ab) anti-IgM, anti-CD40 + Fc und PWM eine zusätzliche Steigerung der Proliferation erzielen. Besonders deutlich zeigte sich der synergistische Effekt von IL-3 in Kombination mit anti-CD40 + Fc, CPG und anti-CD180. Wir konnten keinen eindeutigen Effekt von Interleukin-3 auf die Ausdifferenzierung von naiven B-Lymphozyten zu Memory-B-Zellen oder Plasmazellen nachweisen. Untersuchungen der Expression von CD123 auf mit verschiedenen Substanzen inkubierten B-Zellen ergaben, dass die Stimulanzien CD40 Ligand + aHA und anti-CD180 neben einer Steigerung der B-Zell-Proliferation eine besonders effektive Stimulation der IL-3-Rezeptorexpression erzielen. Unter Stimulation mit CPG oder F(ab)-anti-IgM hingegen konnte ein reduzierter Anteil CD123 positiver Zellen beobachtet werden. Der kostimulatorische Effekt von IL-3 auf die B-Lymphozyten Proliferation fiel bei diesen Stimulanzien gering aus. Auch Interleukin-2 steigert die Expression von CD123 auf B-Lymphozyten. Dabei wirkt IL-2 am ehesten indirekt über eine Aktivierung CD4+ T-Zellen. Interleukin-4 hingegen wirkt hemmend auf die Expression des IL-3-Rezeptors. Interleukin-3 selbst wirkt in Kombination mit den von uns untersuchten Stimulanzien hemmend auf die Expression seines eigenen Rezeptors im Sinne einer negativen Feedbackschleife. Die beschriebenen Wirkungen von Interleukin-3 auf B-Lymphozyten zeigten sich in abgeschwächter Form auch in Anwesenheit von CD4+ Zellen. Da die T-Lymphozyten selbst einen basalen Interleukin-3-Spiegel produzieren ist es nicht verwunderlich, dass die Wirkung des additiven IL-3 einen geringeren Effekt als bei Inkubation mit reinen B-Zellen erzielt. Untersuchungen des von den B-Zellen exprimierten Zytokinprofils zeigten eine Steigerung der IL-6-Ausschüttung unter Stimulation mit IL-3, während die IL-10-Ausschüttung zurückging. Damit spielt Interleukin-3 möglicherweise eine wichtige Rolle in der Regulation der B-Zellantwort bei Entzündungsreaktionen

Cold Atmospheric Plasma Promotes the Immunoreactivity of Granulocytes In Vitro

Cold atmospheric plasma (CAP) reduces bacteria and interacts with tissues and cells, thus improving wound healing. The CAP-related induction of neutrophils was recently described in stained sections of wound tissue in mice. Consequently, this study aimed to examine the functionality of human polymorphonuclear cells (PMN)/granulocytes through either a plasma-treated solution (PTS) or the direct CAP treatment with different plasma modes and treatment durations. PTS analysis yielded mode-dependent differences in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) after CAP treatment. Live-cell imaging did not show any chemo-attractive or NETosis-inducing effect on PMNs treated with PTS. The time to maximum ROS production (TmaxROS) in PMNs was reduced by PTS and direct CAP treatment. PMNs directly treated with CAP showed an altered cell migration dependent on the treatment duration as well as decreased TmaxROS without inducing apoptosis. Additionally, flow cytometry showed enhanced integrin and selectin expression, as a marker of activation, on PMN surfaces. In conclusion, the modification of PMN immunoreactivity may be a main supporting mechanism for CAP-induced improvement in wound healing

Mechanisms of Severe Acute Respiratory Syndrome Coronavirus-Induced Acute Lung Injury

ABSTRACT Systems biology offers considerable promise in uncovering novel pathways by which viruses and other microbial pathogens interact with host signaling and expression networks to mediate disease severity. In this study, we have developed an unbiased modeling approach to identify new pathways and network connections mediating acute lung injury, using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model pathogen. We utilized a time course of matched virologic, pathological, and transcriptomic data within a novel methodological framework that can detect pathway enrichment among key highly connected network genes. This unbiased approach produced a high-priority list of 4 genes in one pathway out of over 3,500 genes that were differentially expressed following SARS-CoV infection. With these data, we predicted that the urokinase and other wound repair pathways would regulate lethal versus sublethal disease following SARS-CoV infection in mice. We validated the importance of the urokinase pathway for SARS-CoV disease severity using genetically defined knockout mice, proteomic correlates of pathway activation, and pathological disease severity. The results of these studies demonstrate that a fine balance exists between host coagulation and fibrinolysin pathways regulating pathological disease outcomes, including diffuse alveolar damage and acute lung injury, following infection with highly pathogenic respiratory viruses, such as SARS-CoV.IMPORTANCESevere acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 and 2003, and infected patients developed an atypical pneumonia, acute lung injury (ALI), and acute respiratory distress syndrome (ARDS) leading to pulmonary fibrosis and death. We identified sets of differentially expressed genes that contribute to ALI and ARDS using lethal and sublethal SARS-CoV infection models. Mathematical prioritization of our gene sets identified the urokinase and extracellular matrix remodeling pathways as the most enriched pathways. By infecting Serpine1-knockout mice, we showed that the urokinase pathway had a significant effect on both lung pathology and overall SARS-CoV pathogenesis. These results demonstrate the effective use of unbiased modeling techniques for identification of high-priority host targets that regulate disease outcomes. Similar transcriptional signatures were noted in 1918 and 2009 H1N1 influenza virus-infected mice, suggesting a common, potentially treatable mechanism in development of virus-induced ALI

Suboptimal Peak Inspiratory Flow and Critical Inhalation Errors are Associated with Higher COPD-Related Healthcare Costs

Purpose: To assess the relationship between suboptimal Peak Inspiratory Flow (sPIF), inhalation technique errors, and non-adherence, with Healthcare Resource Utilisation (HCRU) in Chronic Obstructive Pulmonary Disease (COPD) patients receiving maintenance therapy via a Dry Powder Inhaler (DPI). Patients and methods: The cross-sectional, multi-country PIFotal study included 1434 COPD patients (≥40 years) using a DPI for maintenance therapy. PIF was measured with the In-Check DIAL G16, and sPIF was defined as a typical PIF lower than required for the device. Inhalation technique was assessed by standardised evaluation of video recordings and grouped into 10 steps. Patients completed the "Test of Adherence to Inhalers" questionnaire. HCRU was operationalised as COPD-related costs for primary healthcare, secondary healthcare, medication, and total COPD-related costs in a 1-year period. Results: Participants with sPIF had higher medication costs compared with those with optimal PIF (cost ratio [CR]: 1.07, 95% CI [1.01, 1.14]). Multiple inhalation technique errors were associated with increased HCRU. Specifically, "insufficient inspiratory effort" with higher secondary healthcare costs (CR: 2.20, 95% CI [1.37, 3.54]) and higher total COPD-related costs (CR: 1.16, 95% CI 1.03-1.31). "no breath-hold following the inhalation manoeuvre (<6 s)" with higher medication costs (CR: 1.08, 95% CI [1.02, 1.15]) and total COPD-related costs (CR 1.17, 95% CI [1.07, 1.28]), and "not breathing out calmly after inhalation" with higher medication costs (CR: 1.19, 95% CI [1.04, 1.37]). Non-adherence was not significantly associated with HCRU. Conclusion: sPIF and inhalation technique errors were associated with higher COPD-related healthcare utilisation and costs in COPD patients on DPI maintenance therapy

Predicting the Risk of Rheumatoid Arthritis and Its Age of Onset through Modelling Genetic Risk Variants with Smoking

The improved characterisation of risk factors for rheumatoid arthritis (RA) suggests they could be combined to identify individuals at increased disease risks in whom preventive strategies may be evaluated. We aimed to develop an RA prediction model capable of generating clinically relevant predictive data and to determine if it better predicted younger onset RA (YORA). Our novel modelling approach combined odds ratios for 15 four-digit/10 two-digit HLA-DRB1 alleles, 31 single nucleotide polymorphisms (SNPs) and ever-smoking status in males to determine risk using computer simulation and confidence interval based risk categorisation. Only males were evaluated in our models incorporating smoking as ever-smoking is a significant risk factor for RA in men but not women. We developed multiple models to evaluate each risk factor's impact on prediction. Each model's ability to discriminate anti-citrullinated protein antibody (ACPA)-positive RA from controls was evaluated in two cohorts: Wellcome Trust Case Control Consortium (WTCCC: 1,516 cases; 1,647 controls); UK RA Genetics Group Consortium (UKRAGG: 2,623 cases; 1,500 controls). HLA and smoking provided strongest prediction with good discrimination evidenced by an HLA-smoking model area under the curve (AUC) value of 0.813 in both WTCCC and UKRAGG. SNPs provided minimal prediction (AUC 0.660 WTCCC/0.617 UKRAGG). Whilst high individual risks were identified, with some cases having estimated lifetime risks of 86%, only a minority overall had substantially increased odds for RA. High risks from the HLA model were associated with YORA (P<0.0001); ever-smoking associated with older onset disease. This latter finding suggests smoking's impact on RA risk manifests later in life. Our modelling demonstrates that combining risk factors provides clinically informative RA prediction; additionally HLA and smoking status can be used to predict the risk of younger and older onset RA, respectively

Genetic Studies of Leptin Concentrations Implicate Leptin in the Regulation of Early Adiposity.

Leptin influences food intake by informing the brain about the status of body fat stores. Rare LEP mutations associated with congenital leptin deficiency cause severe early-onset obesity that can be mitigated by administering leptin. However, the role of genetic regulation of leptin in polygenic obesity remains poorly understood. We performed an exome-based analysis in up to 57,232 individuals of diverse ancestries to identify genetic variants that influence adiposity-adjusted leptin concentrations. We identify five novel variants, including four missense variants, in LEP, ZNF800, KLHL31, and ACTL9, and one intergenic variant near KLF14. The missense variant Val94Met (rs17151919) in LEP was common in individuals of African ancestry only, and its association with lower leptin concentrations was specific to this ancestry (P = 2 × 10-16, n = 3,901). Using in vitro analyses, we show that the Met94 allele decreases leptin secretion. We also show that the Met94 allele is associated with higher BMI in young African-ancestry children but not in adults, suggesting that leptin regulates early adiposity

Expression of IL-3 receptors and impact of IL-3 on human T and B cells

A large number of animal models revealed that IL-3 plays an important role for the development of T and B cell-mediated autoimmune diseases. However, little is known about the expression and regulation of IL-3 receptors in human T and B cells and how IL-3 modulates the activation and survival of these cells. We show that the IL-3 receptor CD123 is substantially upregulated on proliferating CD4(+) and CD8(+) T as well as B cells. Upregulation of CD123 differs between various activators and can be further modulated by cytokines. Exposure of human T and B cells to IL-3 enhances proliferation and survival. IL-3 also induces a shift towards secretion of proinflammatory cytokines in T and B cells and reduces the expression of IL-10 in B cells. Thus IL-3 may have proinflammatory and immunostimulatory properties also in human autoimmune diseases

Time course of chemotaxis and chemokinesis of neutrophils following stimulation with IL-8 or FMLP

Polymorphonuclear cells (PMNs) attend to inflammatory sites by chemotactic movement, caused by chemoattractants (CAs) like n-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and interleukin-8 (IL-8). However, distinct but applicable assays for investigations of PMNs’ migration limit in vitro examination. We integrated CD15-bead-based isolation of PMNs with analysing their chemotaxis in a novel 3D-µ-Slide migration chamber. The PMNs were exposed to different concentrations of FMLP and IL-8 (1, 10 and 100 nM) and observed for 180 min in cell-physiological environment conditions. Moving PMNs’ percentage (median and interquartile range) decreased from 62% (27%) to 36% (31%) without CA, from 88% (30%) to 22% (26%) for 1 nM IL-8, from 70% (22%) to 28% (13%) for 100 nM IL-8, from 30% (23%) to 18% (46%) for 1 nM FMLP and from 76% (20%) to 28% (13%) for 100 nM FMLP. Centres of cell movement turned towards the CAs (negative values) within a single 30-min observation period: 5.37 µm (16.82 µm) without CA, −181.37 µm (132.18 µm) with 10 nM and −239.34 µm (152.19 µm) with 100 nM IL-8; −116.2 µm (69.07 µm) with 10 nM and −71.59 µm (98.58 µm) with 100 nM FMLP. FMLP and IL-8 ensure chemotaxis without increase of chemokinesis. 3D-µ-Slide chemotaxis chambers facilitate time course analyses of PMNs’ migration in stable conditions over a long time with concise distinction of chemotaxis and chemokinesis

Olfaction regulates organismal proteostasis and longevity via microRNA-dependent signalling

The maintenance of proteostasis is crucial for any organism to survive and reproduce in an ever-changing environment, but its efficiency declines with age(1). Post-transcriptional regulators such as micrRNAs (miRNAs) control protein translation of target mRNAs, with major consequences for development, physiology and longevity(2,3). Here we show that food odour stimulates organismal proteostasis and promotes longevity in Caenorhabditis elegans through miR-71-mediated inhibition of tir-1 mRNA stability in olfactory AWC neurons. Screening a collection of miRNAs that control ageing(3), we found that the miRNA miR-71 regulates lifespan and promotes ubiquitin-dependent protein turnover, particularly in the intestine. We show that miR-71 directly inhibits the Toll-receptor-domain protein TIR-1 in AWC olfactory neurons and that disruption of miR-71-tir-1 or loss of AWC olfactory neurons eliminates the influence of food source on proteostasis. miR-71-mediated regulation of TIR-1 controls chemotactic behaviour and is regulated by odour. Thus, odour perception influences cell-type-specific miRNA-target interaction, thereby regulating organismal proteostasis and longevity. We anticipate that the proposed mechanism of food perception will stimulate further research on neuroendocrine brain-to-gut communication and may open the possibility for therapeutic interventions to improve proteostasis and organismal health via the sense of smell, with potential implications for obesity, diabetes and ageing