Local Immune Activation and Age Impact on Humoral Immunity in Mice, with a Focus on IgG Sialylation.

Age alters the host's susceptibility to immune induction. Humoral immunity with circulating antibodies, particularly immunoglobulin G (IgG), plays an essential role in immune response. IgG glycosylation in the fragment crystallizable (Fc) region, including sialylation, is important in regulating the effector function by interacting with Fc gamma receptors (FcγRs). Glycosylation is fundamentally changed with age and inflammatory responses. We aimed to explore the regulation of humoral immunity by comparing responses to antigen-induced immune challenges in young and adult mice using a local antigen-induced arthritis mouse model. This study examines the differences in immune response between healthy and immune-challenged states across these groups. Our initial assessment of the arthritis model indicated that adult mice presented more severe knee swelling than their younger counterparts. In contrast, we found that neither histological assessment, bone mineral density, nor the number of osteoclasts differs. Our data revealed an age-associated but not immune challenge increase in total IgG; the only subtype affected by immune challenge was IgG1 and partially IgG3. Interestingly, the sialylation of IgG2b and IgG3 is affected by age and immune challenges but not stimulated further by immune challenges in adult mice. This suggests a shift in IgG towards a pro-inflammatory and potentially pathogenic state with age and inflammation.

Immunomodulatory Nanoparticles for Modulating Arthritis Flares.

Disease-modifying drugs have improved the treatment for autoimmune joint disorders, such as rheumatoid arthritis, but inflammatory flares are a common experience. This work reports the development and application of flare-modulating poly(lactic-co-glycolic acid)-poly(ethylene glycol)-maleimide (PLGA-PEG-MAL)-based nanoparticles conjugated with joint-relevant peptide antigens, aggrecan70-84 and type 2 bovine collagen256-270. Peptide-conjugated PLGA-PEG-MAL nanoparticles encapsulated calcitriol, which acted as an immunoregulatory agent, and were termed calcitriol-loaded nanoparticles (CLNP). CLNP had a ∼200 nm hydrodynamic diameter with a low polydispersity index. In vitro, CLNP induced phenotypic changes in bone marrow derived dendritic cells (DC), reducing the expression of costimulatory and major histocompatibility complex class II molecules, and proinflammatory cytokines. Bulk RNA sequencing of DC showed that CLNP enhanced expression of Ctla4, a gene associated with downregulation of immune responses. In vivo, CLNP accumulated in the proximal lymph nodes after intramuscular injection. Administration of CLNP was not associated with changes in peripheral blood cell numbers or cytokine levels. In the collagen-induced arthritis and SKG mouse models of autoimmune joint disorders, CLNP reduced clinical scores, prevented bone erosion, and preserved cartilage proteoglycan, as assessed by high-resolution microcomputed tomography and histomorphometry analysis. The disease protective effects were associated with increased CTLA-4 expression in joint-localized DC and CD4+ T cells but without generalized suppression of T cell-dependent immune response. The results support the potential of CLNP as modulators of disease flares in autoimmune arthropathies.

Structural and Molecular Changes of Human Chondrocytes Exposed to the Rotating Wall Vessel Bioreactor.

Over the last 30 years, the prevalence of osteoarthritis (OA), a disease characterized by a loss of articular cartilage, has more than doubled worldwide. Patients suffer from pain and progressive loss of joint function. Cartilage is an avascular tissue mostly consisting of extracellular matrix with embedded chondrocytes. As such, it does not regenerate naturally, which makes an early onset of OA prevention and treatment a necessity to sustain the patients' quality of life. In recent years, tissue engineering strategies for the regeneration of cartilage lesions have gained more and more momentum. In this study, we aimed to investigate the scaffold-free 3D cartilage tissue formation under simulated microgravity in the NASA-developed rotating wall vessel (RWV) bioreactor. For this purpose, we cultured both primary human chondrocytes as well as cells from the immortalized line C28/I2 for up to 14 days on the RWV and analyzed tissue morphology, development of apoptosis, and expression of cartilage-specific proteins and genes by histological staining, TUNEL-assays, immunohistochemical detection of collagen species, and quantitative real-time PCR, respectively. We observed spheroid formation in both cell types starting on day 3. After 14 days, constructs from C28/I2 cells had diameters of up to 5 mm, while primary chondrocyte spheroids were slightly smaller with 3 mm. Further inspection of the 14-day-old C28/I2 spheroids revealed a characteristic cartilage morphology with collagen-type 1, -type 2, and -type 10 positivity. Interestingly, these tissues were less susceptible to RWV-induced differential gene expression than those formed from primary chondrocytes, which showed significant changes in the regulation of IL6, ACTB, TUBB, VIM, COL1A1, COL10A1, MMP1, MMP3, MMP13, ITGB1, LAMA1, RUNX3, SOX9, and CASP3 gene expression. These diverging findings might reflect the differences between primary and immortalized cells. Taken together, this study shows that simulated microgravity using the RWV bioreactor is suitable to engineer dense 3D cartilage-like tissue without addition of scaffolds or any other artificial materials. Both primary articular cells and the stable chondrocyte cell line C28/I2 formed 3D neocartilage when exposed for 14 days to an RWV.

The role of calcium crystals and their effect on osteoarthritis pathogenesis.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive degeneration of articular cartilage. Due to its high prevalence and limited treatment options, OA has become one of the most disabling diseases in developed countries. In recent years, OA has been recognized as a heterogenic disease with various phenotypes. Calcium crystal-related endotypes, which are defined by either a distinct functional or pathobiological mechanism, are present in approximately 60% of all OA patients. Two different calcium crystals can accumulate in the joint and thereby calcify the cartilage matrix, which are basic calcium phosphate (BCP) and calcium pyrophosphate (CPP) crystals. The formation of these crystals depends mainly on the balance of phosphate and pyrophosphate, which is regulated by specific proteins controlling the pyrophosphate metabolism. Dysregulation of these molecules subsequently leads to preferential formation of either BCP or CPP crystals. BCP crystals, on the one hand, are directly associated with OA severity and cartilage degradation. They are mostly located in the deeper cartilage layers and are associated with chondrocyte hypertrophy. CPP crystal deposition, on the other hand, is a hallmark of chondrocalcinosis and is associated with aging and chondrocyte senescence. Therefore, BCP and CPP crystals are associated with different chondrocyte phenotypes. However, BCP and CPP crystals are not mutually exclusive and can coexist in OA, creating a mixed endotype of OA. Both crystals clearly play a role in the pathogenesis of OA. However, the exact impact of each crystal type on either driving the disease progression or being a result of chondrocyte differentiation is still to be elucidated.

BCP crystals promote chondrocyte hypertrophic differentiation in OA cartilage by sequestering Wnt3a.

OBJECTIVE: Calcification of cartilage with basic calcium phosphate (BCP) crystals is a common phenomenon during osteoarthritis (OA). It is directly linked to the severity of the disease and known to be associated to hypertrophic differentiation of chondrocytes. One morphogen regulating hypertrophic chondrocyte differentiation is Wnt3a. METHODS: Calcification and sulfation of extracellular matrix of the cartilage was analysed over a time course from 6 to 22 weeks in mice and different OA grades of human cartilage. Wnt3a and ß-catenin was stained in human and murine cartilage. Expression of sulfation modulating enzymes (HS2St1, HS6St1) was analysed using quantitative reverse transcription PCR (RT-PCR). The influence of BCP crystals on the chondrocyte phenotype was investigated using quantitative RT-PCR for the marker genes Axin2, Sox9, Col2, MMP13, ColX and Aggrecan. Using western blot for ß-catenin and pLRP6 we investigated the activation of Wnt signalling. The binding capacity of BCP for Wnt3a was analysed using immunohistochemical staining and western blot. RESULTS: Here, we report that pericellular matrix sulfation is increased in human and murine OA. Wnt3a co-localised with heparan sulfate proteoglycans in the pericellular matrix of chondrocytes in OA cartilage, in which canonical Wnt signalling was activated. In vitro, BCP crystals physically bound to Wnt3a. Interestingly, BCP crystals were sufficient to induce canonical Wnt signalling as assessed by phosphorylation of LRP6 and stabilisation of ß-catenin, and to induce a hypertrophic shift of the chondrocyte phenotype. CONCLUSION: Consequently, our data identify BCP crystals as a concentrating factor for Wnt3a in the pericellular matrix and an inducer of chondrocyte hypertrophy.

Antibody-mediated inhibition of syndecan-4 dimerisation reduces interleukin (IL)-1 receptor trafficking and signalling.

OBJECTIVE: Syndecan-4 (sdc4) is a cell-anchored proteoglycan that consists of a transmembrane core protein and glucosaminoglycan (GAG) side chains. Binding of soluble factors to the GAG chains of sdc4 may result in the dimerisation of sdc4 and the initiation of downstream signalling cascades. However, the question of how sdc4 dimerisation and signalling affects the response of cells to inflammatory stimuli is unknown. METHODS: Sdc4 immunostaining was performed on rheumatoid arthritis (RA) tissue sections. Interleukin (IL)-1 induced extracellular signal-regulated kinases (ERK) phosphorylation and matrix metalloproteinase-3 production was investigated. Il-1 binding to sdc4 was investigated using immunoprecipitation. IL-1 receptor (IL1R1) staining on wild-type, sdc4 and IL1R1 knockout fibroblasts was performed in fluorescence-activated cell sorting analyses. A blocking sdc4 antibody was used to investigate sdc4 dimerisation, IL1R1 expression and the histological paw destruction in the human tumour necrosis factor-alpha transgenic mouse. RESULTS: We show that in fibroblasts, the loss of sdc4 or the antibody-mediated inhibition of sdc4 dimerisation reduces the cell surface expression of the IL-1R and regulates the sensitivity of fibroblasts to IL-1. We demonstrate that IL-1 directly binds to sdc4 and in an IL-1R-independent manner leads to its dimerisation. IL-1-induced dimerisation of sdc4 regulates caveolin vesicle-mediated trafficking of the IL1R1, which in turn determines the responsiveness to IL-1. Administration of antibodies (Ab) against the dimerisation domain of sdc4, thus, strongly reduces the expression IL1R1 on arthritic fibroblasts both in vitro and an animal model of human RA. CONCLUSION: Collectively, our data suggest that Ab that specifically inhibit sdc4 dimerisation may support anti-IL-1 strategies in diseases such as inflammatory arthritis.

Delineating the role of c-FLIP/NEMO interaction in the CD95 network via rational design of molecular probes.

BACKGROUND: Structural homology modeling supported by bioinformatics analysis plays a key role in uncovering new molecular interactions within gene regulatory networks. Here, we have applied this powerful approach to analyze the molecular interactions orchestrating death receptor signaling networks. In particular, we focused on the molecular mechanisms of CD95-mediated NF-κB activation and the role of c-FLIP/NEMO interaction in the induction of this pathway. RESULTS: To this end, we have created the homology model of the c-FLIP/NEMO complex using the reported structure of the v-FLIP/NEMO complex, and rationally designed peptides targeting this complex. The designed peptides were based on the NEMO structure. Strikingly, the experimental in vitro validation demonstrated that the best inhibitory effects on CD95-mediated NF-κB activation are exhibited by the NEMO-derived peptides with the substitution D242Y of NEMO. Furthermore, we have assumed that the c-FLIP/NEMO complex is recruited to the DED filaments formed upon CD95 activation and validated this assumption in silico. Further insight into the function of c-FLIP/NEMO complex was provided by the analysis of evolutionary conservation of interacting regions which demonstrated that this interaction is common in distinct mammalian species. CONCLUSIONS: Taken together, using a combination of bioinformatics and experimental approaches we obtained new insights into CD95-mediated NF-κB activation, providing manifold possibilities for targeting the death receptor network.

Soluble syndecans: biomarkers for diseases and therapeutic options.

Syndecans are important mediators of signalling by transmitting external stimuli into the cells. This role in signal transduction has been attributed mainly to the membrane-bound syndecans. In the last years, however, the soluble ectodomain of syndecans generated by shedding has come into the focus of research as this process has been show to modulate the syndecan-dependent signalling pathways, as well as other pathways. This review summarizes the current knowledge about the induction of syndecan shedding and the different pathways modulated by shed syndecan proteins. This review summarizes the known and putative sheddases for each syndecan and describes the exemplary conditions of sheddase activity for some syndecans. This review summarizes the proposed use of shed syndecans as biomarkers for various diseases, as the shedding process of syndecans depends crucially on tissue- and disease-specific activation of the sheddases. Furthermore, the potential use of soluble syndecans as a therapeutic option is discussed, on the basis of the current literature. LINKED ARTICLES: This article is part of a themed section on Translating the Matrix. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.1/issuetoc.