The soluble CD83 protein prevents bone destruction by inhibiting the formation of osteoclasts and inducing resolution of inflammation in arthritis.

Here we show that soluble CD83 induces the resolution of inflammation in an antigen-induced arthritis (AIA) model. Joint swelling and the arthritis-related expression levels of IL-1ß, IL-6, RANKL, MMP9, and OC-Stamp were strongly reduced, while Foxp3 was induced. In addition, we observed a significant inhibition of TRAP+ osteoclast formation, correlating with the reduced arthritic disease score. In contrast, cell-specific deletion of CD83 in human and murine precursor cells resulted in an enhanced formation of mature osteoclasts. RNA sequencing analyses, comparing sCD83- with mock treated cells, revealed a strong downregulation of osteoclastogenic factors, such as Oc-Stamp, Mmp9 and Nfatc1, Ctsk, and Trap. Concomitantly, transcripts typical for pro-resolving macrophages, e.g., Mrc1/2, Marco, Klf4, and Mertk, were upregulated. Interestingly, members of the metallothionein (MT) family, which have been associated with a reduced arthritic disease severity, were also highly induced by sCD83 in samples derived from RA patients. Finally, we elucidated the sCD83-induced signaling cascade downstream to its binding to the Toll-like receptor 4/(TLR4/MD2) receptor complex using CRISPR/Cas9-induced knockdowns of TLR4/MyD88/TRIF and MTs, revealing that sCD83 acts via the TRIF-signaling cascade. In conclusion, sCD83 represents a promising therapeutic approach to induce the resolution of inflammation and to prevent bone erosion in autoimmune arthritis.

Metabolic reprogramming of osteoclasts represents a therapeutic target during the treatment of osteoporosis.

Osteoclasts are specialised bone resorbing cells that control both physiological and pathological bone turnover. Functional changes in the differentiation and activity of osteoclasts are accompanied by active metabolic reprogramming. However, the biological significance and the in vivo relevance of these events has remained unclear. Here we show that bone resorption of differentiated osteoclasts heavily relies on increased aerobic glycolysis and glycolysis-derived lactate production. While pharmacological inhibition of glycolysis did not affect osteoclast differentiation or viability, it efficiently blocked bone resorption in vitro and in vivo and consequently ameliorated ovariectomy-induced bone loss. Our experiments thus highlight the therapeutic potential of interfering with osteoclast-intrinsic metabolic pathways as possible strategy for the treatment of diseases characterized by accelerated bone loss.

JAK inhibition increases bone mass in steady-state conditions and ameliorates pathological bone loss by stimulating osteoblast function.

Janus kinase (JAK)-mediated cytokine signaling has emerged as an important therapeutic target for the treatment of inflammatory diseases such as rheumatoid arthritis (RA). Accordingly, JAK inhibitors compose a new class of drugs, among which tofacitinib and baricitinib have been approved for the treatment of RA. Periarticular bone erosions contribute considerably to the pathogenesis of RA. However, although the immunomodulatory aspect of JAK inhibition (JAKi) is well defined, the current knowledge of how JAKi influences bone homeostasis is limited. Here, we assessed the effects of the JAK inhibitors tofacitinib and baricitinib on bone phenotype (i) in mice during steady-state conditions or in mice with bone loss induced by (ii) estrogen-deficiency (ovariectomy) or (iii) inflammation (arthritis) to evaluate whether effects of JAKi on bone metabolism require noninflammatory/inflammatory challenge. In all three models, JAKi increased bone mass, consistent with reducing the ratio of receptor activator of NF-κB ligand/osteoprotegerin in serum. In vitro, effects of tofacitinib and baricitinib on osteoclast and osteoblast differentiation were analyzed. JAKi significantly increased osteoblast function (P < 0.05) but showed no direct effects on osteoclasts. Additionally, mRNA sequencing and ingenuity pathway analyses were performed in osteoblasts exposed to JAKi and revealed robust up-regulation of markers for osteoblast function, such as osteocalcin and Wnt signaling. The anabolic effect of JAKi was illustrated by the stabilization of ß-catenin. In humans with RA, JAKi induced bone-anabolic effects as evidenced by repair of arthritic bone erosions. Results support that JAKi is a potent therapeutic tool for increasing osteoblast function and bone formation.

Structural insights into heme binding to IL-36α proinflammatory cytokine.

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.

Full Length Interleukin 33 Aggravates Radiation-Induced Skin Reaction.

The interleukin (IL)-1 family member IL-33 has been described as intracellular alarmin with broad roles in wound healing, skin inflammation but also autoimmunity. Its dichotomy between full length (fl) IL-33 and the mature (m) form of IL-33 and its release by necrosis is still not fully understood. Here, we compare functional consequences of both forms in the skin in vivo, and therefore generated two lines of transgenic mice which selectively overexpress mmIL-33 and flmIL-33 in basal keratinocytes. Transgene mRNA was expressed at high level in skin of both lines but not in organs due to the specific K14 promoter. We could demonstrate that transgenic overexpression of mmIL-33 in murine keratinocytes leads to a spontaneous skin inflammation as opposed to flmIL-33. K14-mmIL-33 mice synthesize and secrete high amounts of mmIL-33 along with massive cutaneous manifestations, like increased epidermis and dermis thickness, infiltration of mast cells in the epidermis and dermis layers and marked hyperkeratosis. Using skin inflammation models such as IL-23 administration, imiquimod treatment, or mechanical irritation did not lead to exacerbated inflammation in the K14-flmIL-33 strain. As radiation induces a strong dermatitis due to apoptosis and necrosis, we determined the effect of fractionated radiation (12 Gy, 4 times). In comparison to wild-type mice, an increase in ear thickness in flmIL-33 transgenic mice was observed 25 days after irradiation. Macroscopic examination showed more severe skin symptoms in irradiated ears compared to controls. In summary, secreted mmIL-33 itself has a potent capacity in skin inflammation whereas fl IL-33 is limited due to its intracellular retention. During tissue damage, fl IL-33 exacerbated radiation-induced skin reaction.

Genome-wide transcriptome analysis of Clavibacter michiganensis subsp. michiganensis grown in xylem mimicking medium.

The interaction between Clavibacter michiganensis subsp. michiganensis with its host, the tomato plant (Solanum lycopersicum), is poorly understood and only few virulence factors are known. While studying of the bacteria in planta is time-consuming and difficult, the analysis in vitro would facilitate research. Therefore, a xylem mimicking medium (XMM) for C. michiganensis subsp. michiganensis was established in this study based on an apoplast medium for Xanthomonas campestris pv. vesicatoria. In contrast to the apoplast medium, XMM contains no sugars, but amino acids which serve as nitrogen and carbon source. As a result, growth in XMM induced transcriptional changes of genes encoding putative sugar, amino acid and iron uptake systems. In summary, mRNA levels of about 8% of all C. michiganensis subsp. michiganensis genes were changed when XMM-grown bacteria were compared to M9 minimal medium-grown cells. Almost no transcriptional changes of genes encoding hydrolytic enzymes were detected, leading to the idea that XMM reflects the situation in the beginning of infection and therefore allows the characterization of virulence factors in this early stage of infection. The addition of the plant wound substance acetosyringone to the XMM medium led to a change in transcript amount, including genes coding for proteins involved in protein transport, iron uptake and regulation processes.

Influence of different hydroxyethyl starch (HES) formulations on fibrinogen measurement in HES-diluted plasma.

BACKGROUND: Fibrinogen is the first coagulation factor becoming critical in dilution coagulopathy. Volume replacement in major blood loss is performed with large volumes of crystalloid and colloid solutions. The latter has been shown to compromise accurate photo-optical measurement of fibrinogen. This study determined the influence of different hydroxyethyl starch (HES) formulations. METHODS: Citrated plasma samples of 8 healthy volunteers were diluted by 30% or 50% with either HES 10% (200/0.5; HES-200), HES 6% (70/0.5; HES-70), or HES 6% (450/0.7; HES-450). Fibrinogen concentrations were determined by photo-optical measurement (Behring coagulation system [BCS]: derived fibrinogen, or Clauss fibrinogen, calibrated for high [CLS] or low fibrinogen concentrations [CLS-low]) as well as mechanical end point determinations (KC4: CLS-KC4). Measured values were compared with calculated values. RESULTS: On average and across all photo-optical methods, fibrinogen concentrations were overestimated, particularly with HES-200. Hydroxyethyl starch-70 and HES-450 did not differ much from each other. Overestimation was relatively greater for 50% dilutions with all HES formulations. Surprisingly, overestimation was most prominent with CLS-low, the method supposed to most reliably measure low fibrinogen concentrations; overestimation amounted to 92% and 120% with HES-200, 54% and 73% with HES-70, and 51% and 79% with HES-450, for 30% and 50% dilutions, respectively. In contrast, CLS-KC4 always yielded sufficiently accurate results. CONCLUSIONS: The study showed that all HES solutions more or less impaired the fibrinogen measurement with the photo-optical method. In particular, overestimation with CLS-low may prevent timely fibrinogen replacement in major blood loss. Hydroxyethyl starch concentration appears to be more relevant for this effect than its molecular size.

Photo-optical methods can lead to clinically relevant overestimation of fibrinogen concentration in plasma diluted with hydroxyethyl starch.

BACKGROUND: Adequate fibrinogen concentration is a crucial component of sufficient perioperative/posttraumatic hemostasis. In major blood loss, large volumes of fluids are being administered, which have been shown to interfere with valid determination of fibrinogen concentration. This may lead to wrong treatment decisions. We studied the variables that cause the discrepancies between measured and true fibrinogen concentrations in samples diluted with volume replacement fluids. METHODS: Citrated plasma samples of healthy volunteers were diluted by 30% and 50% with phosphate buffered saline (PBS), hydroxyethyl starch (HES) 10% (200/0.5), or gelatine (GEL). Fibrinogen concentrations of diluted samples were derived from the prothrombin time (PT) and the Clauss method (CLS) was applied. With the latter, several modifications and combinations of detection principles and thrombin reagents were investigated. Values were compared with ''true,'' that is, calculated values based on the results of undiluted samples for each method. RESULTS: Photo-optical methods resulted in significant overestimation of the fibrinogen concentration in blood diluted with HES, depending on the thrombin reagent used. This was particularly true for modifications of the CLS aimed at measuring low fibrinogen concentrations. Use of another thrombin reagent gave satisfactory results for this modification. The validity of mechanical end point determination methods was considered sufficient and was not influenced by the use of different thrombin reagents. CONCLUSIONS: Fibrinogen determination methods used in situations of major blood loss need to be validated with samples containing significant amounts of volume replacement fluids, particularly colloids. Only some combinations of test principle, detection method, and reagents will give valid results.