Validation of novel multiplex technologies

The parallel analysis of multiple factors, such as cytokines, from small sample size is an interesting approach for assessment of in vivo activation signatures and functionality after ex vivo stimulation. One interesting application is for therapy monitoring, such as safety data, pharmacodynamics, evidences for mode-of-action and side effects, particularly useful for accompanying early phase clinical trials. There are different platforms for Multiplex analysis of ligands available. We compared in this study the performance of three different platforms (Luminex Bio-Plex® 200, MesoScale Discovery®, Ella®) which use different ways of achieving parallel measurements of biomarkers from small liquid sample size. We show examples of in house assessment of intra- and inter-assay variations, determination of range and recovery for classical immunological serum markers and discuss advantages and disadvantages for these three platforms in relation to the question addressed

The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in the p38/TNF-α Pathway of Systemic and Cutaneous Inflammation

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a downstream molecule of p38, involved in the production of TNF-α, a key cytokine, and an established drug target for many inflammatory diseases. We investigated the role of MK2 in skin inflammation to determine its drug target potential. MK2 deficiency significantly decreased plasma TNF-α levels after systemic endotoxin application. Deficient mice showed decreased skin edema formation in chronic 2-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritative dermatitis and in subacute 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity. Surprisingly, MK2 deficiency did not inhibit edema formation in subacute 2,4-dinitrochlorobenzene (DNCB)-induced contact allergy and even increased TNF-α and IL-1β levels as well as granulocyte infiltration in diseased ears. Ear inflammation in this model, however, was inhibited by TNF-α neutralization as it was in the subacute DNFB model. MK2 deficiency also did not show anti-inflammatory effects in acute DNFB-induced contact hypersensitivity, whereas the p38 inhibitor, SB203580, ameliorated skin inflammation supporting a pathophysiological role of p38. When evaluating possible mechanisms, we found that TNF-α production in MK2-deficient spleen cells was strongly diminished after TLR stimulation but less affected after T-cell receptor stimulation. Our data suggest that MK2, in contrast to its downstream effector molecule, TNF-α, has a rather elusive role in T-cell-dependent cutaneous inflammation

Killer-like receptors and GPR56 progressive expression defines cytokine production of human CD4+ memory T cells

All memory T cells mount an accelerated response on antigen reencounter, but significant functional heterogeneity is present within the respective memory T-cell subsets as defined by CCR7 and CD45RA expression, thereby warranting further stratification. Here we show that several surface markers, including KLRB1, KLRG1, GPR56, and KLRF1, help define low, high, or exhausted cytokine producers within human peripheral and intrahepatic CD4+ memory T-cell populations. Highest simultaneous production of TNF and IFN-γ is observed in KLRB1+KLRG1+GPR56+ CD4 T cells. By contrast, KLRF1 expression is associated with T-cell exhaustion and reduced TNF/IFN-γ production. Lastly, TCRβ repertoire analysis and in vitro differentiation support a regulated, progressive expression for these markers during CD4+ memory T-cell differentiation. Our results thus help refine the classification of human memory T cells to provide insights on inflammatory disease progression and immunotherapy development

Cellular and molecular aspects of cytokine regulation

Die Regulation der Expression von Zytokinen und Wachstumsfaktoren mit immunmodulierenden Eigenschaften bleibt ein schwieriges und daher auch sehr interessantes Forschungsgebiet. Wir haben mit den Publikationen, die in dieser Habilitationsschrift zusammengefasst wurden, einige neue Aspekte der Zytokinregulation und ihrer Konsequenzen für die klinische Anwendung beleuchten können. So konnten wir neue zelluläre Effekte des anti- inflammatorischen Zytokins IL-10 beschreiben, indem wir zeigen konnten, dass IL-10 humane Monozyten und Makrophagen vor einer Komplementlyse schützen kann. Außerdem konnten wir demonstrieren, dass IL-10 die Produktion von IFNg in TH1-Zellen unabhängig von der Präsenz von APCs über einen neuen Mechanismus hemmt, aber die IL-17-Produktion in polarisierten TH17-Zellen nicht mehr beeinflussen kann. Dies ist möglicherweise eine der Gründe, warum eine IL-10-Therapie in Erkrankungen mit chronischen Entzündungen wenig Erfolg zeigte. Zur Aufklärung der molekularen Wirkungsweise von IL-10 haben wir eine Reihe von IL-10 regulierten Genen identifiziert, die Gegenstand unser bisherigen und gegenwärtigen Forschungsprojekte sind. Außerdem konnten wir aufgrund von BRET-Untersuchungen ein neues Modell zur Aktivierung von STAT- Molekülen vorschlagen. Die Aktivierung von STAT-Molekülen spielt sowohl für die Regulation der Zytokinantwort als auch für die Generierung von T Zellleukämien eine wichtige Rolle. Unsere Beobachtungen zum molekularen Mechanismus der IL-10 Wirkung haben dazu geführt, dass wir uns näher mit posttranskriptionellen Regulationsmechanismen der Zytokin- und Wachstumsfaktorproduktion beschäftigt haben. Dabei konnten wir zeigen, dass die Regulation der mRNA-Stabilität von Notch-1 über ARE-Bindungsproteine während der Thymozytendifferenzierung entscheidend ist, da das Fehlen zweier ARE-BPs (zfp36l1 und zfp36l2) zu einer unkontrollierten Überexpression von Notch-1 führt und dies die Entwicklung von lymphoblastischen T Zell-Leukämien verursacht. Lymphoblastischen T Zell-Leukämien werden auch durch die fehlgeleitete Expression des Transkriptionsfakors Lmo2 versursacht, die durch retrovirale Gentherapie oder reziproke chromosomale Translokation verursacht sein kann. Wir konnten zeigen, dass Lmo2 während seiner fehlgeleiteten Expression in Thymozyten einen anderen Transkriptionskomplex ausbildet als in seinem physiologischen Zusammenhang der erythroiden Differenzierung. Die dadurch verursachte Fehlregulation von Differenzierungs- und Wachstumsfaktoren führen zu einer Störung des normalen Zelldifferenzierungsprogramms und einer Anhäufung sekundärer Mutationen (u.a. auch im Notch-1 Gen), die zur Entartung der Zelle beitragen. Obwohl wir einige interessante Beiträge zur Zytokinregulation beitragen konnten, sind wir immer noch weit davon entfernt die zugrunde liegenden komplexen molekularen Mechanismen tatsächlich zu verstehen. Daher bleibt die Erforschung dieser Zusammenhänge auch in den nächsten Jahren noch eine große Herausforderung, auf die ich mich freue. Ob das bessere Verständnis der molekularen Grundlagen der Zytokinregulation tatsächlich zu besser wirkenden Therapeutika mit geringerer Nebenwirkung führen wird, kann uns erst die Zukunft zeigen.The expression regulation of cytokines and growth factors remain a difficult but interesting research area. We have contributed with the publications of this cumulative Habilitation to some new knowledge regarding cytokine regulation and its clinical consequences. We could describe some new functions of the anti-inflammatory cytokine IL-10 by demonstrating that it can protect human monocytes and macrophages from complement lysis. Furthermore, we showed that IL-10 can inhibit the IFNg production directly in T cells in the absence of antigen presenting cells but it does not interfere with an established TH17 response, which might be one of the reasons why IL-10 was rather disappointing in clinical trials of treating chronic inflammatory diseases. In order to gain more insight the molecular mechanism of the anti-inflammatory activity of IL-10, we have identified several IL-10 regulated genes in human monocytes. To understand their immunological function has been and is still the focus of my research. Furthermore, we have suggested a new model of STAT3-activation which is a important transcription factor activated IL-10 and other cytokines but also involved in T cell leukaemia. We have analysed here also two different mechanisms of T cell leukaemia generation. Mice lacking two (IL-10 regulated) proteins which bind to AU-rich elements in the mRNA of Notch-1 developed acute T cell leukaemia demonstrating a new role of mRNA regulation in leukaemia. Furthermore we could show that the oncogenic transcription factor Lmo2 is involved in a different transcriptional complex in leukaemia compared to its physiological transcriptional complex in erythroid development. Both mechanisms lead to a disturbed regulation of growth factors and thereby also of cell differentiation programs. Despite the fact that we have contributed a few interesting publications to this field we are far away from understanding the complex molecular details of the regulation of cytokines and growth factors. This remains a difficult but challenging task for me and others in future

Gravitational stress during parabolic flights reduces the number of circulating innate and adaptive leukocyte subsets in human blood.

Gravitational stress occurs during space flights or certain physical activities including extreme sports, where the change in experienced gravitational acceleration can reach large magnitudes. These changes include reduction and increase in the physical forces experienced by the body and may potentially induce pathogenic alterations of physiological processes. The immune system is known to regulate most functions in the human organism and previous studies suggest an impairment of the immune function under gravitational stress. However, systematic studies aiming to investigate the effect of gravitational stress on cellular immune response in humans are lacking. Since parabolic flights are considered as feasible model to investigate a short-term impact of gravitational changes, we evaluated the influence of gravitational stress on the immune system by analyzing leukocyte numbers before and after parabolic flight maneuvers in human blood. To correct for circadian effects, samples were taken at the corresponding time points on ground the day before the flight. The parabolic flight maneuvers led to changes in numbers of different leukocyte subsets. Naïve and memory T and B cell subsets decreased under gravitational stress and lower numbers of basophils and eosinophils were observed. Only circulating neutrophils increased during the parabolic flight. The observed changes could not be attributed to stress-induced cortisol effects, since cortisol levels were not affected. Our data demonstrate that the gravitational stress by parabolic flights can affect all parts of the human immune system. Consequently, it is possible that gravitational stress can have clinically relevant impacts on the control of immune responses

Individual immune cell and cytokine profiles determine platelet-rich plasma composition

Abstract Objective Platelet-rich plasma (PRP) therapy is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune cell and cytokine profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples and assessed the effect of pro-inflammatory cytokines on chondrocytes. Design For each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using whole blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Primary human chondrocyte 2D and 3D in vitro cultures were exposed to recombinant IFN-γ and TNF-α. Proliferation and chondrogenic differentiation were quantitatively assessed. Results All three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood. Furthermore, the pro-inflammatory cytokines IFN-γ and TNF-α were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products. Chondrocyte proliferation was impaired by IFN-γ and enhanced by TNF-α treatment. Differentiation and cartilage formation were compromised upon treatment with both cytokines, resulting in altered messenger ribonucleic acid (mRNA) expression of collagen type 1A1 (COL1A1), COL2A1, and aggrecan (ACAN) as well as reduced proteoglycan content. Conclusions Individuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy, especially as IFN-γ and TNF-α impacted primary chondrocyte proliferation and their characteristic gene expression profile. Both the individual’s immune profile and the concentration method appear to impact the final PRP product. Trial registration This study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4 November 2021 (registration number DRKS00026175)