Anje A. te Velde

The Transcription Factor GATA3 Is Essential for the Function of Human Type 2 Innate Lymphoid Cells

Type 2 innate lymphoid cells (ILC2s) are part of a large family of ILCs that are important effectors in innate immunity, lymphoid organogenesis, and tissue remodeling. ILC2s mediate parasite expulsion but also contribute to airway inflammation, emphasizing the functional similarity between these cells and Th2 cells. Consistent with this, we report that the transcription factor GATA3 was highly expressed by human ILC2s. CRTH2(+) ILC2s were enriched in nasal polyps of patients with chronic rhinosinusitis, a typical type 2-mediated disease. Nasal polyp epithelial cells expressed TSLP, which enhanced STAT5 activation, GATA3 expression, and type 2 cytokine production in ILC2s. Ectopic expression of GATA3 in Lin(-)CD127(+)CRTH2(-) cells resulted in induction of CRTH2 and the capacity to produce high amounts of type 2 cytokines in response to TSLP plus IL-33. Hence, we identify GATA3, potently regulated by TSLP, as an essential transcription factor for the function of human ILC2s.

In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates

BACKGROUND/AIMS The development of custom-made bioreactors for use as a bioartificial liver (BAL) is considered to be one of the last challenges on the road to successful temporary extracorporeal liver support therapy. We devised a novel bioreactor (patent pending) which allows individual perfusion of high density cultured hepatocytes with low diffusional gradients, thereby more closely resembling the conditions in the intact liver lobuli. METHODS The bioreactor consists of a spirally wound nonwoven polyester matrix, i.e. a sheet-shaped, three-dimensional framework for hepatocyte immobilization and aggregation, and of integrated hydrophobic hollow-fiber membranes for decentralized oxygen supply and CO2 removal. Medium (plasma in vivo) was perfused through the extrafiber space and therefore in direct hepatocyte contact. Various parameters were assessed over a period of 4 days including galactose elimination, urea synthesis, lidocaine elimination, lactate/pyruvate ratios, amino acid metabolism, pH, the last day being reserved exclusively for determination of protein secretion. RESULTS Microscopic examination of the hepatocytes revealed cytoarchitectural characteristics as found in vivo. The biochemical performance of the bioreactor remained stable over the investigated period. The urea synthesizing capacity of hepatocytes in the bioreactor was twice that of hepatocytes in monolayer cultures. Flow sensitive magnetic resonance imaging (MRI) revealed that the bioreactor construction ensured medium flow through all parts of the device irrespective of its size. CONCLUSIONS The novel bioreactor showed encouraging efficiency. The device is easy to manufacture with scale-up to the liver mass required for possible short-term support of patients in hepatic failure.

How honey kills bacteria

With the rise in prevalence of antibioticresistant bacteria, honey is increasingly valued for its antibacterial activity. To characterize all bactericidal factors in a medical‐grade honey, we used a novel approach of successive neutralization of individual honey bactericidal factors. All bacteria tested, including Bacillus subtilis, methicillin‐resistant Staphylococcus aureus, extended‐spectrum ß‐lactamase producing Escherichia coli, ciprofloxacin‐resistant Pseudomonas aeruginosa, and vancomycin‐resistant Enterococcus faecium, were killed by 10–20% (v/v) honey, whereas ≥40% (v/v) of a honey‐equivalent sugar solution was required for similar activity. Honey accumulated up to 5.62 ± 0.54 mM H2O2 and contained 0.25 ± 0.01 mM methylglyoxal (MGO). After enzymatic neutralization of these two compounds, honey retained substantial activity. Using B. subtilis for activity‐guided isolation of the additional antimicrobial factors, we discovered bee defensin‐1 in honey. After combined neutralization of H2O2, MGO, and bee defensin‐1, 20% honey had only minimal activity left, and subsequent adjustment of the pH of this honey from 3.3 to 7.0 reduced the activity to that of sugar alone. Activity against all other bacteria tested depended on sugar, H2O2, MGO, and bee defensin‐1. Thus, we fully characterized the antibacterial activity of medical‐grade honey.—Kwakman, P. H. S., te Velde, A. A., de Boer, L., Speijer, D., Vandenbroucke‐Grauls, C. M.J. E., Zaat, S. A.J. How honey kills bacteria. FASEB J. 24, 2576–2582 (2010). www.fasebj.org

Pretreatment with interferon-γ enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis

Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohns disease. MSCs are pluripotent cells with immunosuppressive properties. Recent data suggest that resting MSCs do not have significant immunomodulatory activity, but that the immunosuppressive function of MSCs has to be elicited by interferon‐γ (IFN‐γ). In this article, we assessed the effects of IFN‐γ prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN‐γ and assessed their therapeutic effects in dextran sodium sulfate (DSS)‐ and trinitrobenzene sulfonate (TNBS)‐induced colitis in mice. We found that mice treated with IMSCs (but not MSCs) showed a significantly attenuated development of DSS‐induced colitis. Furthermore, IMSCs alleviated symptoms of TNBS‐induced colitis. IMSC‐treated mice displayed an increase in body weight, lower colitis scores, and better survival rates compared with untreated mice. In addition, serum amyloid A protein levels and local proinflammatory cytokine levels in colonic tissues were significantly suppressed after administration of IMSC. We also observed that IMSCs showed greater migration potential than unstimulated MSCs to sites within the inflamed intestine. In conclusion, we show that prestimulation of MSCs with IFN‐γ enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN‐γ activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo. STEM CELLS 2011;29:1549–1558

Increased expression of DC-SIGN+IL-12+IL-18+ and CD83+IL-12-IL-18- dendritic cell populations in the colonic mucosa of patients with Crohn's disease.

Dentritic cells (DC) as antigen‐presenting cells are most likely responsible for regulation of abnormal T cell activation in Crohns disease (CD), a chronic inflammatory bowel disease. Wehave analyzed the expression of activation and maturation markers on DC in the colon mucosa from patients with CD compared with normal colon, using immunohistochemical techniques. We found two distinct populations of DC present in CD patients: a DC‐specific ICAM‐3 grabbing non‐integrin (DC‐SIGN)+ population that was present scattered throughout the mucosa, and a CD83+ population that was present in aggregated lymphoid nodules and as single cells in the lamina propria. In normal colon the number of DC‐SIGN+ DC was lower and CD83+ DC were detected only in very few solitary lymphoid nodules. Co‐expression of activation markers and cytokine synthesis was analyzed with three‐color confocal laser scanning microscopy analysis. CD80 expression was enhanced on the majority of DC‐SIGN+ DC in CD patients, whereas only a proportion of the CD83+ DC co‐expressed CD80 in CD as well as in normal tissue. Surprisingly, IL‐12 and IL‐18were only detected in DC‐SIGN+ DC and not in CD83+ DC. A similar pattern of cytokine production was observed in normal colon albeit to a much lesser extent. The characteristics ofthese in‐situ‐differentiated DC markedly differ from the in‐vitro‐generated DC that simultaneously express DC‐SIGN, CD83 and cytokines.

Composition of Innate Lymphoid Cell Subsets in the Human Skin: Enrichment of NCR+ ILC3 in Lesional Skin and Blood of Psoriasis Patients

Innate lymphoid cells (ILCs) are increasingly appreciated as important regulators of tissue homeostasis and inflammation. However, their role in human skin remains obscure. We found that healthy peripheral blood CD117(+) ILC3, lacking the natural cytotoxicity receptor (NCR) NKp44 (NCR(-) ILC3), CD117(-)NCR(-)CRTH2(-)CD161(+) ILC1, and CRTH2(+) ILC2, express the skin-homing receptor cutaneous lymphocyte antigen (CLA). NCR(+) ILC3 were scarce in peripheral blood. Consistently, we identified in normal skin ILC2 and NCR(-) ILC3, a small proportion of CD161(+) ILC1, and hardly any NCR(+) ILC3, whereas NCR(+) ILC3 were present in cultured dermal explants. The skin ILC2 and NCR(+) ILC3 subsets produced IL-13 and IL-22, respectively, upon cytokine stimulation. Remarkably, dermal NCR(-) ILC3 converted to NCR(+) ILC3 upon culture in IL-1β plus IL-23, cytokines known to be involved in psoriatic inflammation. In line with this observation, significantly increased proportions of NCR(+) ILC3 were present in lesional skin and peripheral blood of psoriasis patients as compared with skin and blood of healthy individuals, respectively, whereas the proportions of ILC2 and CD161(+) ILC1 remained unchanged. NCR(+) ILC3 from skin and blood of psoriasis patients produced IL-22, which is regarded as a key driver of epidermal thickening, suggesting that NCR(+) ILC3 may participate in psoriasis pathology.

Prediction of antitumour necrosis factor clinical efficacy by real-time visualisation of apoptosis in patients with Crohn’s disease

Background: The human anti-tumour necrosis factor (TNF) antibody infliximab binds to the membrane TNF and subsequently induces apoptosis of activated lamina propria T lymphocytes in patients with Crohn’s disease in vitro. Aim: To test whether the ability of rapid anti-TNF-induced apoptosis in the gut predicts the efficacy of anti-TNF treatment in inflammatory bowel disease. Methods:99mTechnetium–annexin V single-photon emission computer tomography (SPECT) was performed in 2 models of murine experimental colitis and in 14 patients with active Crohn’s disease as assessed by the Crohńs Disease Activity Index (CDAI) to study the effect of anti-TNF treatment on apoptosis in the intestine during active colitis. Disease activity was evaluated 2 weeks after infliximab infusion using the CDAI (definition response: drop of >100 points). Results: Colonic uptake of 99mTc-annexin V significantly increased in 2,4,6-trinitrobenzene sulphonate-induced colitis as well as in transfer colitis on administration of anti-TNF antibodies compared with a control antibody as determined with dedicated animal pinhole SPECT. In addition, uptake of 99mTc–annexin V significantly increased in patients with active Crohn’s disease responding to infliximab treatment. Colonic 99mTc–annexin V uptake ratio (mean (SEM)) increased from 0.24 (0.03) to 0.41(0.07) (p<0.01), 24 h after infliximab infusion (5 mg/kg). A mean increase of 98.7% in colonic uptake of 99mTc–annexin V could be detected in 10 of the 14 responding patients (CDAI >100 points at week 2) compared with 15.2% in non-responding patients (p = 0.03). Analysis of the mucosal biopsy specimens identified lamina propria T cells as target cells undergoing apoptosis. Conclusions: These in vivo observations support the notion that colonic uptake of 99mTc–annexin V correlates with clinical benefit of anti-TNF treatment and might be predictive of therapeutic success.

Comparative analysis of colonic gene expression of three experimental colitis models mimicking inflammatory bowel disease

Background Mouse models of inflammatory bowel diseases (IBD) are used to unravel the pathophysiology of IBD and to study new treatment modalities, but their relationship to Crohns disease (CD) or ulcerative colitis (UC) is speculative. Methods Using Agilent mouse TOX oligonucleotide microarrays, we analyzed colonic gene expression profiles in three widely used models of experimental colitis. In 2 of the models (TNBS and DSS‐induced colitis), exogenous agents induce the colitis. In the third model the colitis is induced after transfer of a T‐cell population (CD4+CD45RBhigh T cells) that lacks regulatory cells into an immunodeficient host. Results Compared with control mice, in DSS, TNBS, and the CD45RB transfer colitis mice, 387, 21, and 582 genes were more than 2‐fold upregulated in the intestinal mucosa. Analyses of exclusively shared gene expression profiles between the different models revealed that DSS/transfer colitis share 69 concordantly upregulated genes, DSS/TNBS 6, and TNBS/transfer colitis 1. Seven genes were upregulated in all three models. The CD45RB transfer model expression profile included the most genes that are known to be upregulated in IBD. Of 32 genes that are known to change transcriptional activity in IBD (TNF, IFN‐&ggr;, Lt&bgr;, IL‐6, IL‐16, IL‐18R1, IL‐22, CCR2, 7, CCL2, 3, 4, 5, 7, 11, 17, 20, CXCR3, CXCL1, 5, 10, Mmp3, 7,9, 14, Timp1, Reg3&ggr;, and Pap, S‐100a8, S‐100a9, Abcb1, and Ptgs2), 2/32 are upregulated in TNBS, 15/32 are upregulated or downregulated in DSS and 30/32 are upregulated or downregulated in the CD45RB transfer colitis. Conclusion The pattern of gene expression in the CD45RB transfer model most closely reflects altered gene expression in IBD. (Inflamm Bowel Dis 2007)

Two Major Medicinal Honeys Have Different Mechanisms of Bactericidal Activity

Honey is increasingly valued for its antibacterial activity, but knowledge regarding the mechanism of action is still incomplete. We assessed the bactericidal activity and mechanism of action of Revamil® source (RS) honey and manuka honey, the sources of two major medical-grade honeys. RS honey killed Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa within 2 hours, whereas manuka honey had such rapid activity only against B. subtilis. After 24 hours of incubation, both honeys killed all tested bacteria, including methicillin-resistant Staphylococcus aureus, but manuka honey retained activity up to higher dilutions than RS honey. Bee defensin-1 and H2O2 were the major factors involved in rapid bactericidal activity of RS honey. These factors were absent in manuka honey, but this honey contained 44-fold higher concentrations of methylglyoxal than RS honey. Methylglyoxal was a major bactericidal factor in manuka honey, but after neutralization of this compound manuka honey retained bactericidal activity due to several unknown factors. RS and manuka honey have highly distinct compositions of bactericidal factors, resulting in large differences in bactericidal activity.

The Epidermal Growth Factor-Seven Transmembrane (EGF-TM7) Receptor CD97 Is Required for Neutrophil Migration and Host Defense

The epidermal growth factor-seven transmembrane (EGF-TM7) family is a group of seven-span transmembrane receptors predominantly expressed by cells of the immune system. Family members CD97, EGF module-containing mucin-like receptor (EMR) 1, EMR2, EMR3, EMR4, and EGF-TM7-latrophilin-related protein are characterized by an extended extracellular region with a variable number of N-terminal EGF-like domains. EGF-TM7 receptors bind cellular ligands as demonstrated by the interaction of CD97 with decay accelerating factor (CD55) and dermatan sulfate. Investigating the effect of newly generated mAb on the migration of neutrophilic granulocytes, we here report for the first time in vivo data on the function of CD97. In dextran sulfate sodium-induced experimental colitis, we show that homing of adoptively transferred neutrophils to the colon was significantly delayed when cells were preincubated with CD97 mAb. The consequences of this defect in neutrophil migration for host defense are demonstrated in a murine model of Streptococcus pneumoniae-induced pneumonia. Mice treated with CD97 mAb to EGF domain 1 (1B2) and EGF domain 3 (1C5) displayed a reduced granulocytic inflammatory infiltrate at 20 h after inoculation. This was associated with a significantly enhanced outgrowth of bacteria in the lungs at 44 h and a strongly diminished survival. Together, these findings indicate an essential role for CD97 in the migration of neutrophils.