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

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

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

Antibodies and IL-3 support helminth-induced basophil expansion

Basophils are powerful mediators of Th2 immunity and are present in increased numbers during allergic inflammation and helminth infection. Despite their ability to potentiate Th2 immunity the mechanisms regulating basophil development remain largely unknown. We have found a unique role for isotype-switched antibodies in promoting helminth-induced basophil production following infection of mice with Heligmosomoides polygyrus bakeri or Nippostrongylus brasiliensis. H. polygyrus bakeri-induced basophil expansion was found to occur within the bone marrow, and to a lesser extent the spleen, and was IL-3 dependent. IL-3 was largely produced by CD4+CD49b+NK1.1− effector T cells at these sites, and required the IL-4Rα chain. However, antibody-deficient mice exhibited defective basophil mobilization despite intact T-cell IL-3 production, and supplementation of mice with immune serum could promote basophilia independently of required IL-4Rα signaling. Helminth-induced eosinophilia was not affected by the deficiency in isotype-switched antibodies, suggesting a direct effect on basophils rather than through priming of Th2 responses. Although normal type 2 immunity occurred in the basopenic mice following primary infection with H. polygyrus bakeri, parasite rejection following challenge infection was impaired. These data reveal a role for isotype-switched antibodies in promoting basophil expansion and effector function following helminth infection

Differential Roles of MAPK Kinases MKK3 and MKK6 in Osteoclastogenesis and Bone Loss

Bone mass is maintained by osteoclasts that resorb bone and osteoblasts that promote matrix deposition and mineralization. Bone homeostasis is altered in chronic inflammation as well as in post-menopausal loss of estrogen, which favors osteoclast activity that leads to osteoporosis. The MAPK p38 alpha is a key regulator of bone loss and p38 inhibitors preserve bone mass by inhibiting osteoclastogenesis. p38 function is regulated by two upstream MAPK kinases, namely MKK3 and MKK6. The goal of this study was to assess the effect of MKK3- or MKK6-deficiency on osteoclastogenesis in vitro and on bone loss in ovariectomy-induced osteoporosis in mice. We demonstrated that MKK3 but not MKK6, regulates osteoclast differentiation from bone marrow cells in vitro. Expression of NFATc1, a master transcription factor in osteoclastogenesis, is decreased in cells lacking MKK3 but not MKK6. Expression of osteoclast-specific genes Cathepsin K, osteoclast-associated receptor and MMP9, was inhibited in MKK3-/- cells. The effect of MKK-deficiency on ovariectomy-induced bone loss was then evaluated in female WT, MKK3-/- and MKK6-/- mice by micro-CT analysis. Bone loss was partially inhibited in MKK3-/- as well as MKK6-/- mice, despite normal osteoclastogenesis in MKK6-/- cells. This correlated with the lower osteoclast numbers in the MKK-deficient ovariectomized mice. These studies suggest that MKK3 and MKK6 differentially regulate bone loss due to estrogen withdrawal. MKK3 directly mediates osteoclastogenesis while MKK6 likely contributes to pro-inflammatory cytokine production that promotes osteoclast formation

Caspase-8 promotes scramblase-mediated phosphatidylserine exposure and fusion of osteoclast precursors

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized

IL-1β Suppresses Innate IL-25 and IL-33 Production and Maintains Helminth Chronicity.

Approximately 2 billion people currently suffer from intestinal helminth infections, which are typically chronic in nature and result in growth retardation, vitamin A deficiency, anemia and poor cognitive function. Such chronicity results from co-evolution between helminths and their mammalian hosts; however, the molecular mechanisms by which these organisms avert immune rejection are not clear. We have found that the natural murine helminth, Heligmosomoides polygyrus bakeri (Hp) elicits the secretion of IL-1β in vivo and in vitro and that this cytokine is critical for shaping a mucosal environment suited to helminth chronicity. Indeed in mice deficient for IL-1β (IL-1β(-/-)), or treated with the soluble IL-1βR antagonist, Anakinra, helminth infection results in enhanced type 2 immunity and accelerated parasite expulsion. IL-1β acts to decrease production of IL-25 and IL-33 at early time points following infection and parasite rejection was determined to require IL-25. Taken together, these data indicate that Hp promotes the release of host-derived IL-1β that suppresses the release of innate cytokines, resulting in suboptimal type 2 immunity and allowing pathogen chronicity

Cell-Intrinsic NF-κB Activation Is Critical for the Development of Natural Regulatory T Cells in Mice

regulatory T (Treg) cells develop in the thymus and represent a mature T cell subpopulation critically involved in maintaining peripheral tolerance. The differentiation of Treg cells in the thymus requires T cell receptor (TCR)/CD28 stimulation along with cytokine-promoted Foxp3 induction. TCR-mediated nuclear factor kappa B (NF-κB) activation seems to be involved in differentiation of Treg cells because deletion of components of the NF-κB signaling pathway, as well as of NF-κB transcription factors, leads to markedly decreased Treg cell numbers in thymus and periphery. thymic Treg precursors and their further differentiation into mature Treg cells. Treg cell development could neither be completely rescued by the addition of exogenous Interleukin 2 (IL-2) nor by the presence of wild-type derived cells in adoptive transfer experiments. However, peripheral NF-κB activation appears to be required for IL-2 production by conventional T cells, thereby participating in Treg cell homeostasis. Moreover, pharmacological NF-κB inhibition via the IκB kinase β (IKKβ) inhibitor AS602868 led to markedly diminished thymic and peripheral Treg cell frequencies.Our results indicate that Treg cell-intrinsic NF-κB activation is essential for thymic Treg cell differentiation, and further suggest pharmacological NF-κB inhibition as a potential therapeutic approach for manipulating this process

Die Rolle von regulatorischen T Zellen im Knochenmetabolismus

The objective of this Ph.D. thesis was to investigate the role of regulatory T cells (Tregs) in physiological and pathological bone remodeling. We first in vitro characterized the suppressive effects of Tregs on the differentiation of the bone resorptive cells or osteoclasts. The effect was found to be directly mediated and to require cell to cell contact between the monocytic precursors of osteoclasts and Tregs. In addition, Tregs mediated expression of TGFβ, IL-4 and IL-10 contributed, but was not essential, to the inhibitory effect on osteoclastogenesis. The co-stimulatory receptor CTLA-4 expressed on the surface of Tregs was the main mediator of their suppressive effect that was abolished in monocytes lacking both the CTLA-4 ligands CD80 and CD86. These in vitro data showing that CD4+CD25+Foxp3+ Tregs suppress osteoclast formation, prompted us to in vivo investigate the role of Tregs in bone homeostasis. We therefore analyzed bone parameters of mice with increased Treg numbers (foxp3-transgenic mice). Indeed foxp3tg mice developed high bone mass and were protected from ovariectomy-induced osteoporosis; inflammatory osteopenia and arthritic bone destruction, whereas foxp3-deficiency enhanced bone loss. Tregs directly protect against bone loss since a significant increased bone mass was observed following adoptive Tregs transfer in RAG1 deficient mice (RAG1-/-). The positive skeletal effects of Tregs were entirely mediated through inhibition of bone resorption by osteoclasts. Binding of Tregs to CD80/CD86 on osteoclast precursors was essential for inhibiting osteoclast differentiation in vivo as shown by the osteopenic phenotype that developed in CD80/86 deficient mice (CD80/86-/-) due to increased osteoclast differentiation. Mechanistically, engagement of CD80/86 by Tregs through CTLA-4 induced IDO expression in osteoclast precursors and thereby increased tryptophan catabolism and apoptosis. These effects were not found in CD80/86 deficient osteoclast precursors. In summary, the presented in vitro and in vivo results demonstrate that Foxp3+ Tregs can actively and directly control bone resorption during physiological and pathological bone remodeling and thus preserve bone mass. Consequently, immune regulation is actively protecting bone mass.Ziel dieser Doktorarbeit war es, zu klären ob und wie regulatorische T Zellen (Tregs) die Osteoklasten Differenzierung in vitro sowie den Knochenstoffwechsel in vivo beeinflussen können. Diese Fragestellungen sollten nicht nur einen tieferen Einblick in die Interaktion von Immunsystem und Knochen erzielen, sondern auch dazu dienen, Strategien zu entdecken, wie das Immunsystem den Knochenabbau verhindert. Dazu wurden CD4+CD25+Foxp3+ T Zellen (Tregs) isoliert und mit CD11b+ Osteoklastenvorläuferzellen kultiviert. Die Osteoklasten Differenzierung wurde mittels einer Tartrat-resistenten sauren Phosphatase (TRAP) Färbung nach 4-6 Tagen in Kultur untersucht. Zudem wurde die Fähigkeit der Osteoklasten zur Knochenresoprtion mittels Resorptionassay auf Osteologic TM Platten analysiert, Transwell-Kulturexperimente durchgeführt und Zytokine blockiert, um die Mechanismen der Interaktion zwischen Tregs und Osteoklasten aufzuklären. Wir konnten zeigen, dass regulatorische CD4+CD25+Foxp3+ T Zellen im Gegensatz zu CD4+CD25-, Foxp3- T Zellen die Osteoklastendifferenzierung durch M-CSF und RANKL dosisabhängig blockieren. Deren resorptive Aktivität war bis zu 80% reduziert sobald Tregs zu Osteoklasten Vorläuferzellen gegeben wurden. Die Blockade der Osteoklastendifferenzierung basierte nicht auf einem Ungleichgewicht zwischen RANKL und OPG Verhältnis, sondern war wesentlich von einem direkten Zell-Zell Kontakt zwischen CTLA-4 auf Tregs und CD80/86 auf Osteoklastenvorläuferzellen abhängig. Die Expression von TGFβ, IL-4 und IL-10 durch Tregs, hatte einen zusätzlichen blockierenden Effekt auf die Osteoklastendiffernzierung, war aber nicht essentiell für die inhibitorische Wirkung von Tregs auf die Osteoklastogenese verantwortlich. Diese ersten in vitro Daten zeigen, dass CD4+CD25+Foxp3+ Tregs die Differenzierung von Osteoklasten aus Osteoklastenvorläuferzellen hemmen können und zeigen eine neue regulierende Beziehung zwischen dem Immunsystem und dem Knochenstoffwechsel. In den folgenden in vivo Versuchen stellten wir uns die Frage ob diese regulierende Beziehung zwischen dem Immunsystem und dem Knochenstoffwechsel auch in vivo nachweisbar ist. Dazu konnten wir zeigen, dass Mäuse mit einem erhöhten Anteil an Tregs (foxp3-transgene Mäuse) eine signifikant erhöhte Knochendichte aufweisen. Zudem waren foxp3-transgene Mäuse sowohl von der postmenopausalen Osteoporose als auch von inflammatorischer Osteopenie und arthritischer Knochendestruktion geschützt. Tregs erhöhten zudem die Knochenmasse in Lymphozyten-defiziente RAG1-/- Mäuse (RAG1-/-), wobei nach adoptiven Transfer von Treg eine signifikant höhere Knochendichte zu beobachten war. Die beschriebenen positiven Effekte der Tregs auf die systemische Knochendichte wurden durch die Hemmung der Knochenresoption durch Osteoklasten verursacht. Der direkte Kontakt von Tregs mit CD80/86 auf Osteoklastenverläuferzellen war essentiell für die Hemmung der Knochenresoption durch Osteoklasten. In Übereinstimmung damit zeigen CD80/86 knock-out Mäuse (CD80/86-/-) einen osteoporotischen Phänotyp basierend auf einer erhöhten Anzahl an Osteoklasten. Die Binding von CD80/86 durch CTLA-4 auf Tregs induziert in Osteoklastenvorläuferzellen die Expression von IDO und somit einen verstärkten Tryptophan Stoffwechsel was dazu führt das Osteoklastenvorläuferzellen vermehrt apoptotisch werden und sich somit nicht in reife knochenresorbierende Osteoklasten differenzieren können. Osteoklastenvorläuferzellen aus CD80/86-/- Mäusen zeigen keine Hochregulierung der IDO mRNA Expression und werden nach Kontakt mit CTLA-4 Ig oder Tregs auch nicht apoptotisch. Zusammenfassend kann man sagen dass die hier präsentierten in vitro sowie in vivo Daten zeigen, dass Tregs direkt die Knochenresorption sowohl im physiologischen als auch im pathologischen Knochenstoffwechsel kontrollieren und somit eine neue wichtige Verbindung zwischen dem Immunsystem und dem Knochenstoffwechsel darstellen