Kristina Thamm

IFN-γ Production by Allogeneic Foxp3+ Regulatory T Cells Is Essential for Preventing Experimental Graft-versus-Host Disease

It is emerging that CD4+Foxp3+ regulatory T (Treg) cells can produce the proinflammatory cytokine IFN-γ when stimulated in a Th1 cytokine environment. In this study, we report that Foxp3+ Treg cells readily produced IFN-γ in vivo in a highly inflammatory model of graft-versus-host disease (GVHD) and during a Th1-dominated immune response to intracellular bacteria. Moreover, stimulation in vitro via TCR in the presence of IL-12 alone was sufficient to induce IFN-γ production by Treg cells in a dose-dependent manner. Transfer of donor Treg cells can prevent lethal GVHD; therefore, we used this model as a robust readout for in vivo Treg function. Interestingly, >50% of allogeneic donor, but not residual recipient Foxp3+ Treg cells produced IFN-γ after transplantation, suggesting that this cytokine production was alloantigen specific. These IFN-γ producers were stable Foxp3+ Treg cells because methylation analysis of the Foxp3 gene locus of transferred and reisolated Treg cells during GVHD showed a fully demethylated Treg-specific–demethylated region. Next, we addressed whether IFN-γ production was supporting or rather impairing the immunosuppressive function of Treg cells during GVHD. Blocking of IFN-γ with specific mAb completely abolished the beneficial effect of donor Treg cells. We could further show that only wild-type Treg cells, but not Treg cells from IFN-γ–deficient donor mice, prevented GVHD. This indicated that Treg cell-intrinsic IFN-γ production was required for their protective function. In conclusion, our data show that IFN-γ produced by Foxp3+ Treg cells has essential immune-regulatory functions that are required for prevention of experimental GVHD.

Lung-targeted RNA interference against angiopoietin-2 ameliorates multiple organ dysfunction and death in sepsis.

Objective:Angiopoietin-2, a protein secreted by stimulated endothelium and an antagonist of the endothelium-stabilizing receptor Tie2, contributes to the pathophysiology of septic multiple organ dysfunction. We tested the therapeutic potential of a pulmonary-endothelium-specific RNA interference–based angiopoietin-2 targeting strategy in sepsis. Design:Laboratory and animal research. Settings:Research laboratories of the Medical School Hannover, Department of Nephrology and Hypertension, Hannover and Silence Therapeutics GmbH, Berlin. Subjects:C57Bl/6 mice. Interventions:Lung-endothelium-specific angiopoietin-2 small interfering RNA was administered both before and after sepsis induction (cecal ligation and puncture or lipopolysaccharides) intravenously. Measurements and Main Results:Angiopoietin-2 small interfering RNA was highly specific and reduced angiopoietin-2 expression in the septic murine lungs up to 73.8% (p = 0.01) and enhanced the phosphorylation of Tie2 both in control and septic animals. Angiopoietin-2 small interfering RNA reduced pulmonary interleukin-6 transcription, intercellular adhesion molecule expression, neutrophil infiltration, and vascular leakage. Manifestations of sepsis were also attenuated in distant organs, including the kidney, where renal function was improved without affecting local angiopoietin-2 production. Finally, angiopoietin-2 small interfering RNA ameliorated the severity of illness and improved survival in cecal ligation and puncture, both as a pretreatment and as a rescue intervention. Conclusion:The Tie2 antagonist angiopoietin-2 represents a promising target against sepsis-associated multiple organ dysfunction. A novel RNA interference therapeutic approach targeting gene expression in the pulmonary endothelium could be a clinically relevant pharmacological strategy to reduce injurious angiopoietin-2 synthesis.

Drug Repurposing Screen Identifies Foxo1-Dependent Angiopoietin-2 Regulation in Sepsis.

Objective:The recent withdrawal of a targeted sepsis therapy has diminished pharmaceutical enthusiasm for developing novel drugs for the treatment of sepsis. Angiopoietin-2 is an endothelial-derived protein that potentiates vascular inflammation and leakage and may be involved in sepsis pathogenesis. We screened approved compounds for putative inhibitors of angiopoietin-2 production and investigated underlying molecular mechanisms. Design:Laboratory and animal research plus prospective placebo-controlled randomized controlled trial (NCT00529139) and retrospective analysis (NCT00676897). Setting:Research laboratories of Hannover Medical School and Harvard Medical School. Patients:Septic patients/C57Bl/6 mice and human endothelial cells. Interventions:Food and Drug Administration–approved library screening. Measurements and Main Results:In a cell-based screen of more than 650 Food and Drug Administration–approved compounds, we identified multiple members of the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor drug class (referred to as statins) that suppressed angiopoietin-2. Simvastatin inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase, which in turn activated PI3K-kinase. Downstream of this signaling, PI3K-dependent phosphorylation of the transcription factor Foxo1 at key amino acids inhibited its ability to shuttle to the nucleus and bind cis-elements in the angiopoietin-2 promoter. In septic mice, transient inhibition of angiopoietin-2 expression by liposomal siRNA in vivo improved absolute survival by 50%. Simvastatin had a similar effect, but the combination of angiopoietin-2 siRNA and simvastatin showed no additive benefit. To verify the link between statins and angiopoietin-2 in humans, we performed a pilot matched case-control study and a small randomized placebo-controlled trial demonstrating beneficial effects on angiopoietin-2. Conclusions:3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors may operate through a novel Foxo1-angiopoietin-2 mechanism to suppress de novo production of angiopoietin-2 and thereby ameliorate manifestations of sepsis. Given angiopoietin-2’s dual role as a biomarker and candidate disease mediator, early serum angiopoietin-2 measurement may serve as a stratification tool for future trials of drugs targeting vascular leakage.

Effect of extracorporeal cytokine removal on vascular barrier function in a septic shock patient

BackgroundSepsis and septic shock are major healthcare problems, affecting millions of individuals around the world each year. Pathophysiologically, septic multiple organ dysfunction (MOD) is a life-threatening condition caused by an overwhelming systemic inflammatory response of the host’s organism to an infection. We experimentally tested if high circulating cytokine levels might increase vascular permeability—a critical hallmark of the disease—and if this phenomenon can be reversed by therapeutic cytokine removal (CytoSorb®) in an exemplary patient.Case presentationA 32-year-old Caucasian female presented with septic shock and accompanying acute kidney injury (Sequential Organ Failure Assessment (SOFA) = 18) to our ICU. In spite of a broad anti-infective regimen, adequate fluid resuscitation, and high doses of inotropics and catecholamines, she remained refractory hypotensive. The extraordinary severity of septic shock suggested an immense overwhelming host response assumingly accompanied by a notable cytokine storm such as known from patients with toxic shock syndrome. Thus, a CytoSorb® filter was added to the dialysis circuit to remove excess shock-perpetuating cytokines. To analyze the endothelial phenotype in vitro before and after extracorporeal cytokine removal, we tested the septic shock patient’s serum on human umbilical vein endothelial cells (HUVECs). The effect on endothelial integrity was assessed both on the morphological (fluorescent immunocytochemistry for VE-cadherin and F-actin) and functional (transendothelial electrical resistance (TER)) level that was recorded in real time with an “electric cell-substrate impedance sensing” (ECIS) system (ibidi). We found (1) severe alterations of cell-cell contacts and the cytoskeletal architecture and (2) profound functional permeability changes, the putative cellular correlate of the clinical vascular leakage syndrome. However, the endothelial barrier was protected from these profound adverse effects when HUVECs were challenged with septic shock serum that was collected after extracorporeal cytokine removal.ConclusionsBeneficial observations of extracorporeal cytokine removal in septic shock patients might—at least in part—be promoted via protection of vascular barrier function.

Angiopoietin-2 and biliary diseases: elevated serum, but not bile levels are associated with cholangiocarcinoma.

Background The diagnosis of cholangiocarcinoma (CC) is challenging especially in patients with primary sclerosing cholangitis (PSC) and often delayed due to the lack of reliable markers. Angiopoietin-2 (Angpt-2) has been employed as a biomarker of angiogenesis and might be involved in tumor neoangiogenesis. Aim To evaluate the diagnostic potential of Angpt-2 as a biomarker to detect patients with CC. Methods Bile and serum Angpt-2 levels were measured in patients with CC (n = 45), PSC (n = 74), CC complicating PSC (CC/PSC) (n = 11) and patients with bile duct stones (n = 37) in a cross sectional study. Diagnostic accuracy of Angpt-2 was compared to carbohydrate antigen 19-9 (CA19-9). Fluorescent immunohistochemistry from human CC liver tissue samples was performed to localize the origin of Angpt-2. Results Serum Angpt-2 concentration was significantly elevated in patients with CC compared to control patients (p<0.05). Diagnostic accuracy of Angpt-2 as determined by receiver operating characteristic (ROC) analysis resulted in a higher area under the curve (AUC) value compared to CA19-9 (AUC: 0.85 versus 0.77; 95% confidence interval (CI): 0.74–0.93 versus 0.65–0.87, respectively). Angpt-2 was also detectable in bile, but was not associated with the presence of CC. Immunohistochemistry revealed a strong induction of Angpt-2 expression in the tumor vasculature. Conclusions Circulating Angpt-2 in serum might be a promising protein candidate locally derived from the tumor vasculature in patients with CC. Measurement of Angpt-2 in serum may be useful for diagnosis and further clinical management of patients with CC.

Donor Vδ1 + γδ T cells expand after allogeneic hematopoietic stem cell transplantation and show reactivity against CMV-infected cells but not against progressing B-CLL

Abstractγδ T lymphocytes play an important role in immune reactions towards infections and malignancies. In particular, Vγ9–Vδ1+ T lymphocytes are thought to play protective antiviral roles in human CMV infection. Recently, Vδ1+ T lymphocytes were proposed to also have anti- B-CLL reactivity. Here we report a case of 48-year-old man who received allogeneic stem cell transplantation for progressive B-CLL. Within one year after transplantation, lymphoma relapsed despite a dramatic increase of Vδ1+ T cells in the patient’s blood. In vitro killing assays revealed activity of patient’s γδ cells against CMV target cells, but not against the relapsing lymphoma-cells. This argues for a contribution of Vδ1+ cells in the immune reaction against CMV reactivation, but does not support a strong correlation of expanded Vδ1+ T cells and favorable disease outcome in B-CLL patients.

Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to an infection leading to systemic inflammation and endothelial barrier breakdown. The vascular-destabilizing factor Angiopoietin-2 (Angpt-2) has been implicated in these processes in humans. Here we screened in an unbiased approach FDA-approved compounds with respect to Angpt-2 suppression in endothelial cells (ECs) in vitro. We identified Flunarizine – a well-known anti-migraine calcium channel (CC) blocker – being able to diminish intracellular Angpt-2 protein in a time- and dose-dependent fashion thereby indirectly reducing the released protein. Moreover, Flunarizine protected ECs from TNFα-induced increase in Angpt-2 transcription and vascular barrier breakdown. Mechanistically, we could exclude canonical Tie2 signalling being responsible but found that three structurally distinct T-type - but not L-type - CC blockers can suppress Angpt-2. Most importantly, experimental increase in intracellular calcium abolished Flunarizine’s effect. Flunarizine was also able to block the injurious increase of Angpt-2 in murine endotoxemia in vivo. This resulted in reduced pulmonary adhesion molecule expression (intercellular adhesion molecule-1) and tissue infiltration of inflammatory cells (Gr-1). Our finding could have therapeutic implications as side effects of Flunarizine are low and specific sepsis therapeutics that target the dysregulated host response are highly desirable.

Pharmacological Tie2 activation in kidney transplantation

AIM To investigate the therapeutic potential of vasculotide (VT) - a Tie2 activating therapeutic - in kidney transplantation. METHODS We performed a murine MHC-mismatched renal transplant model (C57Bl/6 male into Balb/c female) with 60 min cold and 30 min warm ischemia time. 500 ng VT was administered i.p. to donor mice 1 h before organ removal. In addition, recipients received 500 ng VT i.p. directly and 3 d after surgery. Survival was monitored and remaining animals were sacrificed 28 d after transplantation. In this model, we analyzed: (1) organ function; (2) Kaplan-Meier survival; (3) organ damage (periodic acid Schiff staining) via semi-quantitative scoring [0-4 (0 = no injury/inflammation to 4 = very severe injury/inflammation)]; (4) expression of renal endothelial adhesion molecules (ICAM-1) via immunofluorescence (IF) staining, immunoblotting and qPCR; (5) infiltration of inflammatory cells (IF Gr-1, F4/80); and (6) fibrosis via staining of α-smooth muscle actin (αSMA), Sirius red staining and immunoblotting of SMAD3 activation. RESULTS Exogenous activation of Tie2 with VT resulted in diminished expression of peritubular and glomerular endothelial adhesion molecules. Consequently, infiltration of inflammatory cells (analyzed as ICAM-1, Gr-1 and F4/80 positive cells) was reduced in VT-treated mice compared to controls. Additionally, VT was protective against fibrogenesis after kidney transplantation. Trends towards lower serum creatinine (vehicle: 142 ± 17 μmol/L vs VT: 94 ± 23 μmol/L), urea (vehicle: 76 ± 5 mmol/L vs VT: 60 ± 8 mmol/L) and lactate dehydrogenase (vehicle: 1288 ± 383 iU vs VT: 870 ± 275 iU) were observed on day 6 after transplantation. Kaplan-Meier survival analysis showed improved survival rates in the VT-treated mice that did not reach statistical significance (27% vs 54%, P = 0.24, n = 11 per group). Exogenous activation of Tie2 via VT might reduce infiltration of inflammatory cells into renal tissue thereby protecting the transplant from early graft dysfunction potentially affecting long-term function. CONCLUSION Protection of the endothelial microvasculature via the Tie2 axis in the early transplant setting might hold promise as a therapeutic target.

Role of angiopoietin-2 in infection - A double-edged sword?

The endothelial angiopoietin (Angpt)/Tie2 ligand receptor system maintains vascular quiescence and modulates the response to injury. Angpt-1 is considered the natural Tie2 agonist and receptor ligation leads to its phosphorylation inducing various protective downstream pathways. The natural antagonist - Angpt-2 - appears to inhibit these protective effects. In sepsis, the balance between both ligands is shifted in favor for Angpt-2 and the vasculature is highly dysfunctional, activated and leaky. Circulating levels of Angpt-2 strongly predict mortality in septic patients. Consistently, experimental strategies that target Angpt-2 (e.g. antibody, RNAi, etc.) can protect the vascular barrier and improve survival. However, in vitro is has also been shown that Angpt-2 can act as a dose-dependent Tie2 agonist/antagonist. Based on this, people have wondered if Angpt-2 is per se injurious or if it might have protective effects dependent on the scenario. A recent paper by Safioleas and colleagues showed survival benefits after a therapeutic injection of recombinant Angpt-2 in experimental pyelonephritis. Here, we discuss their counter-intuitive but interesting findings and put them into a global context with respect to the existent literature in the angiopoietin/Tie2 sepsis field.

Angiopoietin-2 in sepsis: A “thera-gnostic” tool to tailor treatments?

We appreciate Zhang et al.’s [1] commentary placing our recent study [2] in the context of how angiogenesis factors may modulate the sepsis phenotype. We share their enthusiasm that Angiopoietin-2 (Angpt-2) represents a compelling target for future investigation, and we appreciate their interest in RNA interference (RNAi) as a viable therapeutic approach. The commentary from Zhang et al. also offers an opportunity to address three points for further consideration. First, pre-formed Angpt-2 protein is stored in endothelial cells and is thought to be released into the circulation by early events of systemic inflammation—e.g., induction of TNFα. In turn, released Angpt-2 protein then potentiates the inflammatory response of the endothelium. However, this paradigm fails to explain why the sickest individuals often exhibit a progressive elevation of circulating Angpt-2 as their clinical status deteriorates over days toward death [3]. In this light, our recent study provides first-in-kind evidence that sustained biosynthesis of Angpt-2, not simply release of pre-formed protein, is itself pathological in sepsis. Angpt-2-directed RNAi reduced mortality whether it was administered before or after the induction of experimental sepsis. This new observation also stresses the need to investigate mechanisms that regulate ANGPT2 gene expression in inflammation. Second, heterogeneity—both between affected individuals and within the time course experienced by a single patient—continues to confound efforts to develop novel treatments in sepsis. Personalized treatments may thus be highly desirable. Angpt-2 concentration is easily measured in the blood, and its elevation is strongly associated with other measures of vascular leakage and with hard outcomes such as the future risk of shock or death [3]. Measurement of circulating Angpt-2 may thus be a promising approach for identifying septic patients whose molecular pathophysiology is driven by vascular leakage. More broadly, Angpt-2 measurements may be used in future trials not only as an inclusion criterion, but also as a means to follow response to therapies targeting the vasculature (whether or not such therapies directly target Angpt-2 itself). Lastly, Zhang et al. introduce the concept of a dual-axis vascular therapy, suggesting that combined inhibition of Angpt-2 and VEGF signaling may yield benefits beyond either approach applied singly. While pre-clinical studies offer encouragement regarding such an approach [4], one might worry about the emergence of vascular toxicity from compound approaches as well [5]. A careful assessment of the risks and benefits is highly desirable.