RANKL-Induced Btn2a2 – A T Cell Immunomodulatory Molecule – During Osteoclast Differentiation Fine-Tunes Bone Resorption

Butyrophilins, which are members of the extended B7 family of immunoregulators structurally related to the B7 family, have diverse functions on immune cells as co-stimulatory and co-inhibitory molecules. Despite recent advances in the understanding on butyrophilins’ role on adaptive immune cells during infectious or autoimmune diseases, nothing is known about their role in bone homeostasis. Here, we analyzed the role of one specific butyrophilin, namely Btn2a2, as we have recently shown that Btn2a2 is expressed on the monocyte/macrophage lineage that also gives rise to bone degrading osteoclasts. We found that expression of Btn2a2 on monocytes and pre-osteoclasts is upregulated by the receptor activator of nuclear factor κ-B ligand (RANKL), an essential protein required for osteoclast formation. Interestingly, in Btn2a2-deficient osteoclasts, typical osteoclast marker genes (Nfatc1, cathepsin K, TRAP, and RANK) were downregulated following RANKL stimulation. In vitro osteoclast assays resulted in decreased TRAP positive osteoclast numbers in Btn2a2-deficient cells. However, Btn2a2-deficient osteoclasts revealed abnormal fusion processes shown by their increased size. In vivo steady state µCT and histological analysis of bone architecture in complete Btn2a2-deficient mice showed differences in bone parameters further highlighting the fine-tuning effect of BTN2a2. Moreover, in rheumatoid arthritis patients and experimental arthritis, we detected significantly decreased serum levels of the secreted soluble Btn2a2 protein. Taken together, we identified the involvement of the immunomodulatory molecule Btn2a2 in osteoclast differentiation with potential future implications in basic and translational osteoimmunology

The Role of Dietary Fiber in Rheumatoid Arthritis Patients: A Feasibility Study

Short-chain fatty acids are microbial metabolites that have been shown to be key regulators of the gut–joint axis in animal models. In humans, microbial dysbiosis was observed in rheumatoid arthritis (RA) patients as well as in those at-risk to develop RA, and is thought to be an environmental trigger for the development of clinical disease. At the same time, diet has a proven impact on maintaining intestinal microbial homeostasis. Given this association, we performed a feasibility study in RA patients using high-fiber dietary supplementation with the objective to restore microbial homeostasis and promote the secretion of beneficial immunomodulatory microbial metabolites. RA patients (n = 36) under routine care received daily high-fiber bars or cereals for 28 days. Clinical assessments and laboratory analysis of immune parameters in blood and stool samples from RA patients were done before and after the high-fiber dietary supplementation. We observed an increase in circulating regulatory T cell numbers, favorable Th1/Th17 ratios, as well as decreased markers of bone erosion in RA patients after 28 days of dietary intervention. Furthermore, patient-related outcomes of RA improved. Based on these results, we conclude that controlled clinical studies of high-fiber dietary interventions could be a viable approach to supplement or complement current pharmacological treatment strategies

The Intestinal Microbiota Contributes to the Ability of Helminths to Modulate Allergic Inflammation

We thank Manuel Kulagin for technical help, Pierre Bonnaventure for portal vein blood sampling, Francisco Sepulveda for technical assistance in GS-MS acquisition, and Dorothee Hahne (Metabolomics Australia, University of Western Australia) for human samples SCFA isolation, acquisition, and analysis. We also thank Cristina Cartoni (Phenotyping Unit, EPFL) for Milliplex analysis, Jessica Dessimoz and her team from the Histology Core Facility (EPFL), Miguel Garcia and his team from the Flow Cytometry Core Facility (EPFL), and staff from the EPFL CPG animal house for excellent animal care. The computations were partially performed at the Vital-IT Center for high-performance computing of the SIB Swiss Institute of Bioinformatics (http://www.vital-it.ch). The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. 310948. Funding for A.W.W. and a subset of the 16S rRNA gene sequencing was provided by the Wellcome Trust (grant number WT 098051). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Btn2a2 Regulates ILC2–T Cell Cross Talk in Type 2 Immune Responses

Innate lymphoid cells (ILC) not only are responsible for shaping the innate immune response but also actively modulate T cell responses. However, the molecular processes regulating ILC-T cell interaction are not yet completely understood. The protein butyrophilin 2a2 (Btn2a2), a co-stimulatory molecule first identified on antigen-presenting cells, has a pivotal role in the maintenance of T cell homeostasis, but the main effector cell and the respective ligands remain elusive. We analyzed the role of Btn2a2 in the ILC-T cell cross talk. We found that the expression of Btn2a2 is upregulated in ILC2 following stimulation with IL-33/IL-25/TSLP. In vitro and in vivo experiments indicated that lack of Btn2a2 expression on ILC2 resulted in elevated T cell responses. We observed an enhanced proliferation of T cells as well as increased secretion of the type 2 cytokines IL-4/IL-5/IL-13 following cocultures with Btn2a2-deficient ILC2. In vivo transfer experiments confirmed the regulatory role of Btn2a2 on ILC2 as Btn2a2-deficient ILC2 induced stronger T cell responses and prevented chronic helminth infections. Taken together, we identified Btn2a2 as a significant player in the regulation of ILC2–T cell interactions

Free Fatty Acids in Bone Pathophysiology of Rheumatic Diseases

Obesity—in which free fatty acid (FFA) levels are chronically elevated—is a known risk factor for different rheumatic diseases, and obese patients are more likely to develop osteoarthritis (OA) also in non-weight-bearing joints. These findings suggest that FFA may also play a role in inflammation-related joint damage and bone loss in rheumatoid arthritis (RA) and OA. Therefore, the objective of this study was to analyze if and how FFA influence cells of bone metabolism in rheumatic diseases. When stimulated with FFA, osteoblasts from RA and OA patients secreted higher amounts of the proinflammatory cytokine interleukin (IL)-6 and the chemokines IL-8, growth-related oncogene α, and monocyte chemotactic protein 1. Receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin, and osteoblast differentiation markers were not influenced by FFA. Mineralization activity of osteoblasts correlated inversely with the level of FFA-induced IL-6 secretion. Expression of the Wnt signaling molecules, axin-2 and β-catenin, was not changed by palmitic acid (PA) or linoleic acid (LA), suggesting no involvement of the Wnt signaling pathway in FFA signaling for osteoblasts. On the other hand, Toll-like receptor 4 blockade significantly reduced PA-induced IL-8 secretion by osteoblasts, while blocking Toll-like receptor 2 had no effect. In osteoclasts, IL-8 secretion was enhanced by PA and LA particularly at the earliest time point of differentiation. Differences were observed between the responses of RA and OA osteoclasts. FFA might therefore represent a new molecular factor by which adipose tissue contributes to subchondral bone damage in RA and OA. In this context, their mechanisms of action appear to be dependent on inflammation and innate immune system rather than Wnt-RANKL pathways

Die Rolle der Galektine in der Clearance und Immunogenität apoptotischer und sekundär nekrotischer Zellen

The objective of this Ph.D. thesis was to investigate the role of galectins in clearance and immunogenicity of apoptotic and secondary necrotic cells We first monitored the potential LPS contamination of galectin preparations. We observed that almost all recombinant produced galectins were contaminated with LPS. We then tested a convenient and effective method for decontaminating the galectins. This was important to continue with further functional assays. To investigate the role of galectins during clearance, we first had to determine the binding characteristics of galectins to viable, apoptotic, secondary apoptotic ad necrotic cells. We observed that galectins display an inhomogeneous binding pattern with an increase of binding towards late stages of apoptosis. Since galectins differentially recognize apoptotic cells they may play a potentially non-redundant role in the recognition and opsonization of apoptotic cells for phagocytic clearance. This assumption was supported by the findings that opsonization of apoptotic cells with galectins increased the rate of phagocytic uptake. Furthermore, opsonization with galectins of apoptotic and secondary necrotic cells was able to shift and decrease the respective immune response, indicating that galectins may act as opsonins for dying and dead cells and play a pivotal role in directing the outcome of phagocytosis. Furthermore, pre-incubation of irradiated tumor cells with galectins decreased or even abrogated the protection from tumor development induced by tumor vaccination. This further underlined our assumption that galectins act as pivoting opsonins for dying and dead cells capable of interfering with the clearance process. Anti-galectin autoantibodies were present in sera of patients with the autoimmune diseases SLE and RA. Since the development of SLE is connected to deficiencies in clearance, the presence of anti-galectin autoantibodies may play a role in the pathogenicity of this autoimmune disease. Further examination of the functional role of galectins may reveal new aspects within the surveillance system to protect against auto-inflammation. In summary, the presented results demonstrate that galectins can actively influence the recognition and clearance of dying and dead cells and thus play a role in the inflammatory outcome of apoptotic cells uptake.Das Ziel dieser Doktorarbeit war es, die Rolle der Galektine in der Clearance und Immunogenität apoptotischer und sekundär nekrotischer Zellen zu untersuchen. Zu Beginn untersuchten wir die potentielle Kontamination der Galektin Präparationen mit LPS. Wir beobachteten, dass fast alle der rekombinant hergestellten Galektin Präparationen mit LPS kontaminiert waren. Daraufhin haben wir eine praktische und effektive Methode für die Dekontaminierung der Galektine getestet, mit der wir die Galektine für funktionelle Untersuchungen reinigen konnten. Um die Rolle der Galektine während der Clearance zu untersuchen, mussten wir zuerst die Bindung an vitale, apoptotische und nekrotische Zellen charakterisieren. Die Galektine zeigten ein in-homogenes Bindungsprofil mit der Tendenz zu einer verstärkten Bindung an spät-apoptotische Zellen. Da die Galektine apoptotische Zellen unterschiedlich stark binden, können sie eine Rolle bei der Erkennung und Opsonisierung apoptotischer Zellen für die Aufnahme durch Phagozyten spielen. Diese Annahme wurde untermauert durch die Ergebnisse, dass Opsonisierung von apoptotischen Zellen mit Galektinen deren Aufnahme durch Phagozyten erhöhte. Darüber hinaus war die Opsonisierung apoptotischer sowie sekundär-nekrotischer Zellen mit Galektine in der Lage, die Immunantwort zu verändern bzw. abzumildern. Diese Erkenntnisse deuten daraufhin, dass die Galektine als Opsonisierungsmoleküle für tote und sterbende Zellen eine zentrale Rolle spielen und in der Lage sind, das Ergebnis der Aufnahme zu beeinflussen. In einem Tumorvakzinierungsmodell konnte auch gezeigt werden, dass Prä-Inkubation mit Galektinen von bestrahlten, apoptotischen Tumorzellen in der Lage war, den Schutz, hervorgerufen durch die Vakzinierung, zu verringern oder ganz abzuschaffen. Dieses Ergebnis bestärkt die Annahme, dass Galektine als Opsonisierungsmoleküle in der Lage sind, den Clearance-Prozess zu beeinflussen. Anti-Galektin Autoantikörper wurden in Seren von Patienten mit den Autoimmunkrankheiten SLE und RA gefunden. Die Entstehung von SLE ist verbunden mit Defekten in der Clearance, und daher ist es möglich, dass anti-Galektin Autoantikörper an der Pathogenität dieser Krankheit beteiligt sind. Eine weitere Untersuchung der funktionellen Rolle der Galektine kann neue Aspekte aufdecken im Überwachungssystem zum Schutz vor Auto-Inflammation und vor Entstehung von Autoimmunkrankheiten. Zusammenfassend kann man sagen, dass die hier präsentierten Ergebnisse eine Rolle der Galektine bei der Erkennung und Clearance toter und sterbender Zellen aufzeigen und dass die Galektine in der Lage sind, die Immunantwort auf die Aufnahme apoptotischer Zellen beeinflussen können

Permanent sex inversion in 1-year-old juveniles of the protogynous dusky grouper (Epinephelus marginatus) using controlled-release 17α-methyltestosterone implants

Long-term treatments (April-June 2004) with 17α-methyltestosterone (MT) and/or human chorionic gonadotropin (hCG) were used to induce precocious sex change in 1-year-old juveniles (mean ± SD body weight of 131 ± 25 g) of the protogynous dusky grouper (Epinephelus marginatus). The MT treatment was given once a month using controlled-release implants (1.5 mg per implant, ∼11 mg kg- 1 body weight), whereas hCG was injected intramuscularly once a month (MT/hCG group) or every 2 weeks (hCG group). In vitro mean (± SEM) daily and total MT release from the implants was 22.6 ± 5.4 μg day- 1 implant- 1 and 686 ± 113 μg implant- 1, respectively. In vivo, plasma MT levels of European sea bas (Dicentrarchus labrax) used as model fish (203 ± 41 g body weight) and given a single MT implant were 18.5 ± 0.5 ng ml- 1 one day after implantation, declined to 9.6 ± 2.7 ng ml- 1 by day 11 and remained stable between 7.9 and 5.5 ng ml- 1 until day 32. Plasma MT levels were significantly elevated (2-way ANOVA, P 0.05) between MT/hCG-treated and control fish were observed in plasma testosterone (T) or 11-keto T (11-KT). Pituitary luteinizing hormone (LH) content was significantly lower in both the MT/hCG and hCG groups, compared to controls (2-way ANOVA, P < 0.001), while plasma LH was below detection limits in all treatments. At the onset of the experiment all but one fish were already differentiated into females, having ovaries with primary oocytes. No signs of vitellogenesis or sex inversion were observed in fish from the control and hCG groups during the study. On the contrary, the MT implants induced complete sex change after 12 weeks, with the gonads organized in lobules and cysts filled with germ cells at all stages of spermatogenesis, including spermatozoa. This sex inversion was permanent and fish underwent complete spermatogenesis again during the following year. The results demonstrate that the produced MT-implants, but not the hCG injections, were highly efficient in inducing both sex inversion and complete spermatogenesis in pre-pubertal dusky grouper at the completion of their second year in life. Production of male fish at such an early age may dramatically reduce by many years the time required for natural puberty, sex inversion and the production of sperm by dusky grouper reared under culture conditions

Inflammatory arthritis and systemic bone loss are attenuated by gastrointestinal helminth parasites

Infections with different helminth species have been observed to ameliorate a variety of chronic inflammatory diseases. Herein, we show that the natural murine helminth species, Heligmosomoides polygyrus bakeri (Hp) is capable of attenuating disease severity in two different inflammatory arthritis models. Furthermore, we show that excretory-secretory (ES) products from Hp directly suppress osteoclast differentiation in vitro. Taken together, these results demonstrate that helminth infections can dampen autoimmune diseases and highlight a previously unrecognized and important role for ES products, by directly impacting on bone destruction

Von Märkten, Konsum und einer besseren Welt. Nachhaltigkeit und Fairtrade im öffentlichen Sektor

Sarter EK, Sack D. Von Märkten, Konsum und einer besseren Welt. Nachhaltigkeit und Fairtrade im öffentlichen Sektor. In: Jantke K, Lottermoser F, Reinhardt J, Rothe D, Stöver J, eds. Nachhaltiger Konsum. Institutionen, Instrumente, Initiativen. Baden-Baden: Nomos Verlagsgesellschaft; 2016: 379-394

Dietary Short-Term Fiber Interventions in Arthritis Patients Increase Systemic SCFA Levels and Regulate Inflammation.

Chronic inflammatory diseases are often initiated and guided by the release of proinflammatory mediators. Rheumatoid arthritis (RA) is caused by an imbalance between the pro- and anti-inflammatory mediators in the joints, thereby favoring chronic inflammation and joint damage. Here, we investigate if short-term high-fiber dietary intervention shifts this towards anti-inflammatory mediators. Healthy controls (n = 10) and RA patients (n = 29) under routine care received daily high-fiber bars for 15 or 30 days, respectively. Stool and sera were analyzed for pro- and anti-inflammatory mediators. A high-fiber dietary intervention resulted in increased anti-inflammatory short-chain fatty acids (SCFA), decreased proarthritic cytokine concentrations, along with a durable shift in the Firmicutes-to-Bacteroidetes ratio. Together, these results further strengthen high-fiber dietary interventions as a practical approach complementing existing pharmacological therapies