Jens G. Gerwien

TNF-α blockade induces IL-10 expression in human CD4+ T cells

IL-17+ CD4+ T (Th17) cells contribute to the pathogenesis of several human inflammatory diseases. Here we demonstrate that TNF-inhibitor (TNFi) drugs induce the anti-inflammatory cytokine IL-10 in CD4+ T cells including IL-17+ CD4+ T cells. TNFi-mediated induction of IL-10 in IL-17+ CD4+ T cells is Treg/Foxp3 independent, requires IL-10 and is overcome by IL-1β. TNFi-exposed IL-17+ CD4+ T cells are molecularly and functionally distinct, with a unique gene signature characterised by expression of IL10 and IKZF3 (encoding Aiolos). We show that Aiolos binds conserved regions in the IL10 locus in IL-17+ CD4+ T cells. Furthermore, IKZF3 and IL10 expression levels correlate in primary CD4+ T cells and Aiolos overexpression is sufficient to drive IL10 in these cells. Our data demonstrate that TNF-α blockade induces IL-10 in CD4+ T cells including Th17 cells and suggest a role for the transcription factor Aiolos in the regulation of IL-10 in CD4+ T cells.

High-dose erythropoietin alters platelet reactivity and bleeding time in rodents in contrast to the neuroprotective variant carbamyl-erythropoietin (CEPO)

The haematopoietic hormone erythropoietin (EPO) has neuroprotective properties and is currently being explored for treatment of stroke and other neurological disorders. Short-term, high-dose treatment with EPO seems to improve neurological function of stroke patients but may be associated with increased thrombotic risk, whereas alternative non-erythropoietic neuroprotective derivatives of EPO, such as carbamylated EPO (CEPO), may be devoid of such side-effects. We investigated the effects of short-term, high-dose treatment with EPO and CEPO on platelet function and haemostasis in healthy mice and rats. Animals received three daily doses of EPO or CEPO (50 microg/kg), and blood was compared with respect to alterations in haematology and platelet reactivity. In rats, treatment with EPO increased the haematocrit to >50% and the mean platelet volume by 37%, while CEPO had no effect on these parameters. Platelets from EPO-treated rats showed an increased sensitivity to thrombin receptor agonist peptides and elevated plasma levels of soluble P-selectin (sP-selectin) were found in treated mice. Further indicators of platelet hyperreactivity in EPO, but not CEPO-treated animals, were significantly increased aggregatory responses to collagen in whole blood and platelet-rich plasma (PRP). The increased platelet reactivity was paralleled by a decreased bleeding time after tail transection in rats. Samples from EPO-treated rats showed an attenuated response to ADP in whole blood aggregometry and thrombelastography (TEG) platelet mapping but not in apyrase-treated PRP, suggesting involvement of ADP receptor desensitization. These findings suggest that while EPO affects various aspects of platelet function, CEPO is devoid of such effects.

A Single-Cell Gene-Expression Profile Reveals Inter-Cellular Heterogeneity within Human Monocyte Subsets.

Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16, and unique gene expression profile. So far, the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study, the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers, and proteins involved in immune regulation. Within the three human monocyte subsets, our descriptive findings show multimodal expression of key immune response genes, such as CD40, NFⱪB1, RELA, TLR4, TLR8 and TLR9. Furthermore, we discovered one subgroup of cells within the classical monocytes, which showed alterations of 22 genes e.g. IRF8, CD40, CSF1R, NFⱪB1, RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes, respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation, independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance, and can be useful in unravelling inter-cellular variation in leukocyte populations, identifying subpopulations involved in disease pathogenesis and help tailor new therapies.

Phenotypic, Functional, and Gene Expression Profiling of Peripheral CD45RA+ and CD45RO+ CD4+CD25+CD127(low) Treg Cells in Patients With Chronic Rheumatoid Arthritis.

Conflicting evidence exists regarding the suppressive capacity of Treg cells in the peripheral blood (PB) of patients with rheumatoid arthritis (RA). The aim of this study was to determine whether Treg cells are intrinsically defective in RA.

Phenotypic, functional and gene expression profiling of peripheral CD45RA+ and CD45RO+ CD4+CD25+CD127low regulatory T cells in rheumatoid arthritis

Conflicting evidence exists regarding the suppressive capacity of Treg cells in the peripheral blood (PB) of patients with rheumatoid arthritis (RA). The aim of this study was to determine whether Treg cells are intrinsically defective in RA.

NKG2D ligand expression in Crohn's disease and NKG2D-dependent stimulation of CD8+ T cell migration

Interaction between the activating NKG2D receptor on lymphocytes and its ligands MICA, MICB, and ULBP1-6 modulate T and NK cell activity and may contribute to the pathogenesis of Crohns disease (CD). NKG2D ligands are generally not expressed on the cell surface of normal, non-stressed cells, but expression of MICA and MICB in CD intestine has been reported. In this exploratory study, we further characterize the expression of NKG2D and its ligands, including the less well-described ULBP4-6, in CD, and test if NKG2D ligand interactions are involved in the migration of activated T cells into the affected mucosal compartments. Intestinal tissue from CD patients and healthy controls were analyzed by flow cytometry, mass cytometry, and immunohistochemistry for expression of NKG2D and ligands, and for cytokine release. Furthermore, NKG2D-dependent chemotaxis of activated CD8+ T cells across a monolayer of ligand-expressing human intestinal endothelial cells was examined. Activated lymphocytes down-regulated NKG2D expression upon accumulation in inflamed CD intestine. NKG2D expression on CD56+ T and γδ T cells from inflamed tissue seemed inversely correlated with CRP levels and cytokine release. B cells, monocytes, mucosal epithelium, and vascular endothelium expressed NKG2D ligands in inflamed CD intestine. The expression of NKG2D ligands was correlated with cytokine release, but was highly variable between patients. Stimulation of vascular intestinal endothelial cells in vitro induced expression of NKG2D ligands, including MICA/B and ULBP2/6. Blockade of NKG2D on CD8+ T cells inhibited the migration over ligand-expressing endothelial cells. Intestinal induction of NKG2D ligands and ligand-induced down-regulation of NKG2D in CD suggest that the NKG2D-ligand interaction may be involved in both the activation and recruitment of NKG2D+ lymphocytes into the inflamed CD intestine.

The soluble cytoplasmic tail of CD45 (ct-CD45) in human plasma contributes to keep T cells in a quiescent state.

The cytoplasmic tail of CD45 (ct‐CD45) is proteolytically cleaved and released upon activation of human phagocytes. It acts on T cells as an inhibitory, cytokine‐like factor in vitro. Here, we show that ct‐CD45 is abundant in human peripheral blood plasma from healthy adults compared with plasma derived from umbilical cord blood and plasma from patients with rheumatoid arthritis or systemic lupus erythematosus. Plasma depleted of ct‐CD45 enhanced T‐cell proliferation, while addition of exogenous ct‐CD45 protein inhibited proliferation and reduced cytokine production of human T lymphocytes in response to TCR signaling. Inhibition of T‐cell proliferation by ct‐CD45 was overcome by costimulation via CD28. T‐cell activation in the presence of ct‐CD45 was associated with an upregulation of the quiescence factors Schlafen family member 12 (SLFN12) and Krueppel‐like factor 2 (KLF2) as well as of the cyclin‐dependent kinase (CDK) inhibitor p27kip1. In contrast, positive regulators of the cell cycle such as cyclin D2 and D3 as well as CDK2 and CDK4 were found to be downregulated in response to ct‐CD45. In summary, we demonstrate that ct‐CD45 is present in human plasma and sets the threshold of T‐cell activation.

The protease inhibitor cystatin C down‐regulates the release of IL‐β and TNF‐α in lipopolysaccharide activated monocytes

Human cystatin C, a member of the cysteine proteinase‐inhibitory family, is produced by all nucleated cells and has important roles in regulating natural immunity. Nematode homologs to human cystatin C have been shown to have anti‐inflammatory effects on monocytes and to reduce colitis in mice. In Crohns disease, pathogenic activated monocytes help drive inflammatory processes via the release of proinflammatory cytokines and chemokines. In particular, tumor necrosis factor‐α–producing inflammatory monocytes have a central role in the intestinal inflammation in patients with Crohns disease. We investigated the potential of human cystatin C to regulate pathogenic activated monocytes and its potential as an Immunomodulator in Crohns disease. We found that cystatin C significantly decreased the lipopolysaccharide‐stimulated release and expression of interleukin‐1β and tumor necrosis factor‐α in monocyte and peripheral blood mononuclear cell cultures from healthy donors, whereas interleukin‐6 and interleukin‐8 levels were unchanged. A similar reduction of interleukin‐1β and tumor necrosis factor‐α was also seen in peripheral blood mononuclear cell cultures from patients with Crohns disease, and in particular, tumor necrosis factor‐α was reduced in supernatants from lamina propria cell cultures from patients with Crohns disease. Further investigation revealed that cystatin C was internalized by monocytes via an active endocytic process, decreased phosphorylation of the mitogen‐activated protein kinase pathway extracellular signal‐regulated kinase‐1/2, and altered surface marker expression. The ability of cystatin C to modulate the cytokine expression of monocytes, together with its protease‐inhibitory function, indicates that modulation of the local cystatin C expression could be an option in future Crohns disease therapy.

TNF-inhibitor drugs regulate human pathogenic Th17 cells through induction of IL-10

Results Ex vivo analysis of patients with RA on TNFi therapy revealed an enrichment of Th17 cells in peripheral blood compared to those on disease-modifying anti-rheumatic drugs or healthy controls. However, we also found an increase in IL-10-producing CD4+ T-cells. The enrichment in IL-17+ and IL-10+ CD4+ T-cells, including IL17+IL-10+ co-expressing CD4+ T-cells, was recapitulated in vitro by the addition of TNFi drugs (adalimumab, infliximab, etanercept, and certolizumab) to human monocyte/CD4+ T-cell co-cultures. IL-10 induction was independent of FcgR binding, IL-10 and CD4+CD25+ Tregs. TNFi-induced Th17 cells were functionally distinct as shown by an ability to modulate CD14+ monocytes in an IL-10-dependent manner. We report the identification of a transcription factor that is strongly associated with IL-10 expression in TNFi-induced IL-17+ CD4+ T-cells, and show that overexpression of this transcription factor drives IL-10 expression in primary CD4+ T-cells.

Engagement of distinct epitopes on CD43 induces different co‐stimulatory pathways in human T cells

Co‐receptors, being either co‐stimulatory or co‐inhibitory, play a pivotal role in T‐cell immunity. Several studies have indicated that CD43, one of the abundant T‐cell surface glycoproteins, acts not only as a potent co‐receptor but also as a negative regulator for T‐cell activation. Here we demonstrate that co‐stimulation of human peripheral blood (PB) T cells through two distinct CD43 epitopes recognized by monoclonal antibodies (mAb) CD43‐6E5 (T6E5‐act) and CD43‐10G7 (T10G7‐act) potently induced T‐cell proliferation. However, T‐cell co‐stimulation through two CD43 epitopes differentially regulated activation of nuclear factor of activated T cells (NFAT) and nuclear factor‐κB (NF‐κB) transcription factors, T‐cell cytokine production and effector function. T6E5‐act produced high levels of interleukin‐22 (IL‐22) and interferon‐γ (IFN‐γ) similar to T cells activated via CD28 (TCD28‐act), whereas T10G7‐act produced low levels of inflammatory cytokines but higher levels of regulatory cytokines transforming growth factor‐β (TGF‐β) and interleukin‐35 (IL‐35). Compared with T6E5‐act or to TCD28‐act, T10G7‐act performed poorly in response to re‐stimulation and further acquired a T‐cell suppressive function. T10G7‐act did not directly inhibit proliferation of responder T cells, but formed stable heterotypic clusters with dendritic cells (DC) via CD2 to constrain activation of responder T cells. Together, our data demonstrate that CD43 is a unique and polarizing regulator of T‐cell function.