Low-Dose Radiotherapy Leads to a Systemic Anti-Inflammatory Shift in the Pre-Clinical K/BxN Serum Transfer Model and Reduces Osteoarthritic Pain in Patients

Osteoarthritis (OA) is the leading degenerative joint disease in the western world and leads, if left untreated, to a progressive deterioration of joint functionality, ultimately reducing quality of life. Recent data has shown, that especially OA of the ankle and foot are among the most frequently affected regions. Current research in OA points towards a complex involvement of various cell and tissue types, often accompanied by inflammation. Low-dose radiotherapy (LDRT) is widely used for the treatment of degenerative and inflammatory diseases. While the reported analgesic effects are well known, the underlying molecular mechanisms are only poorly understood. We therefore correlated a clinical approach, looking at pain reduction in 196 patients treated with LDRT with a pre-clinical approach, utilizing the K/BxN serum transfer mouse model using flow cytometry and multiplex ELISA for analysis. While an improvement of symptoms in the majority of patients was found, patients suffering from symptoms within the tarsi transversa show a significantly lower level of improvement. Further, a significant impact of therapy success was detected depending on whether only one or both feet were affected. Further, patients of younger age showed a significantly better outcome than older ones while needing fewer treatment series. When looking on a cellular level within the mouse model, a systemic alteration of immune cells namely a shift from CD8+ to CD4+ T cells and reduced numbers of DCs was observed. A general reduction of inflammatory cytokines was detected, with significant alterations in IL-4 and IL-17 levels, all of which could potentially be responsible for the highly effective clinical improvement in patients. Taken together our data indicate that LDRT can be regarded as a highly effective treatment option for patients suffering from OA of the foot and ankle, in terms of analgesic effects, especially in younger patients. Furthermore, the observed effects are mediated by an interplay of cellular and soluble immune factors, as observed in the K/BxN serum transfer model. With this interdisciplinary approach we aim to encourage the usage of LDRT as an additive treatment strategy not only as a last resort, but also earlier in the course of disease.

Can fracture healing be accelerated by serum transfer in head trauma cases? An experimental head trauma model in rats

Objectives: In this study, we aimed to investigate whether the positive union effect caused by head trauma could be transferred between individuals. Materials and methods: Seventy-two male rats with an average weight of 375 g were used in this study and divided into four groups including 18 in each group. Group 1 consisted of serum donor rats that were exposed to head trauma, while Group 2 consisted of study rats with long bone fractures that were given the serum obtained from the rats in Group 1, Group 3 included control rats with isolated long bone fractures, and Group 4 included control rats with both head trauma and long bone fractures. For radiological evaluation, the ratio of the width of the callus to the width of the neighboring diaphysis was considered as the callus-to- diaphyseal ratio in the study and control groups. Histopathological and radiological evaluations was made on Days 10, 20, and 30. Results: In evaluation of the radiological data regarding the callus- to-diaphyseal ratio, Group 3 was found to have significantly lower radiological values than Group 4 on Day 10 (p=0.006). Group 2 had significantly higher values than Group 3 (p=0.02). On Day 20, Group 2 exhibited significantly higher radiological values than Group 3 (p=0.004), but lower than Group 4 (p=0.032). As for Day 30, Group 2 exhibited significantly higher radiological values than Group 3, but lower than Group 4 (p=0.001). In the evaluation of the Huo scores obtained for histopathological evaluation, there was no significant difference among the groups on Days 10, 20, and 30 (p=0.295, p=0.569, and p=0.729, respectively). Conclusion: Our study results suggest that the osteoinductive effect after head trauma can be transmitted between individuals by means of serum transfer.

Fetal Macrophages Exposed to Salmonella Antigens Elicit Protective Immunity Against Overwhelming Salmonella Challenge in A Murine Model

Despite the evidence for fetal immunization following maternal infection, it remained a mystery how the fetal immune system was primed by vertically-transmitted pathogens or microbial antigens, especially before its full maturation. We previously demonstrated the capacity of fetal macrophages for endocytosing oncoprotein and allergens to bridge towards adaptive immunity in postnatal life. To investigate the immunological consequences of fetal contact with microbial antigens and the role of fetal macrophages in the defense against infection before T-cell development, we exposed gestational day 14 murine fetuses and their macrophages to flagellin and heat-killed Salmonella Typhimurium. Recipients with in utero exposure to Salmonella antigens or adoptive transfer of microbial antigen-loaded fetal macrophages were examined for immune responses to Salmonella antigens and resistance to virulent Salmonella challenge. Fetal exposure to microbial antigens or adoptive transfer of microbial antigen-loaded fetal macrophages could confer antigen-specific adaptive immunity. However, protective immunity against lethal Salmonella challenge was only granted to those receiving heat-killed Salmonella antigens, presenting as heightened recall responses of serum anti-lipopolysaccharide immunoglobulins and interferon-gamma. In immunized recipients surviving Salmonella challenge, their serum transfer to succeeding recipients provided immediate protection from lethal Salmonella challenge in preference to lymphocyte transfer, indicating a more active role of humoral immunity in the prevention of Salmonella invasiveness. Our study sheds insight on the role of fetal macrophages in immunogenicity to transplacental pathogens regardless of fetal lymphocyte maturity, paving the way for fetal macrophage therapies to enhance vaccine responsiveness or increase resistance to pathogenic microorganisms in perinatal life.

Neuraminidase Inhibitor Zanamivir Ameliorates Collagen-Induced Arthritis

Altered sialylation patterns play a role in chronic autoimmune diseases such as rheumatoid arthritis (RA). Recent studies have shown the pro-inflammatory activities of immunoglobulins (Igs) with desialylated sugar moieties. The role of neuraminidases (NEUs), enzymes which are responsible for the cleavage of terminal sialic acids (SA) from sialoglycoconjugates, is not fully understood in RA. We investigated the impact of zanamivir, an inhibitor of the influenza virus neuraminidase, and mammalian NEU2/3 on clinical outcomes in experimental arthritides studies. The severity of arthritis was monitored and IgG titers were measured by ELISA. (2,6)-linked SA was determined on IgG by ELISA and on cell surfaces by flow cytometry. Zanamivir at a dose of 100 mg/kg (zana-100) significantly ameliorated collagen-induced arthritis (CIA), whereas zana-100 was ineffective in serum transfer-induced arthritis. Systemic zana-100 treatment reduced the number of splenic CD138+/TACI+ plasma cells and CD19+ B cells, which was associated with lower IgG levels and an increased sialylation status of IgG compared to controls. Our data reveal the contribution of NEU2/3 in CIA. Zanamivir down-modulated the T and B cell-dependent humoral immune response and induced an anti-inflammatory milieu by inhibiting sialic acid degradation. We suggest that neuraminidases might represent a promising therapeutic target for RA and possibly also for other antibody-mediated autoimmune diseases.

Neuronal interleukin-1 receptors mediate pain in chronic inflammatory diseases

Chronic pain is a major comorbidity of chronic inflammatory diseases. Here, we report that the cytokine IL-1β, which is abundantly produced during multiple sclerosis (MS), arthritis (RA), and osteoarthritis (OA) both in humans and in animal models, drives pain associated with these diseases. We found that the type 1 IL-1 receptor (IL-1R1) is highly expressed in the mouse and human by a subpopulation of TRPV1+ dorsal root ganglion neurons specialized in detecting painful stimuli, termed nociceptors. Strikingly, deletion of the Il1r1 gene specifically in TRPV1+ nociceptors prevented the development of mechanical allodynia without affecting clinical signs and disease progression in mice with experimental autoimmune encephalomyelitis and K/BxN serum transfer–induced RA. Conditional restoration of IL-1R1 expression in nociceptors of IL-1R1–knockout mice induced pain behavior but did not affect joint damage in monosodium iodoacetate–induced OA. Collectively, these data reveal that neuronal IL-1R1 signaling mediates pain, uncovering the potential benefit of anti–IL-1 therapies for pain management in patients with chronic inflammatory diseases.

LB0002 A FIRST IN CLASS THERAPEUTIC NANOPARTICLE FOR SPECIFIC TARGETING OF ANTI-CITRULLINATED PROTEIN ANTIBODY AMELIORATES SERUM TRANSFER AND COLLAGEN INDUCED ARTHRITIS

Background:Rheumatoid arthritis (RA) is an immune mediated inflammatory disease with autoimmune features, including antibodies to citrullinated proteins and peptides (ACPAs). Several in vitro studies have suggested a pathogenic role of ACPAs in RA. However, in vivo proof of this concept has been hampered by the lack of therapeutic strategies to reduce or deplete ACPA in serum and synovial fluid. Previously, we constructed a chitosan-hyaluronic acid nanoparticle formulation with the ability to use neutrophil recruitment as a delivery mechanism to inflamed joints. Specifically, nanoparticles got phagocytosed and then released to synovial fluid upon death of the short-lived neutrophilsObjectives:We hypothesized that reducing ACPA levels would have a therapeutic effect by blocking cytokine production. In this study, we prepared and tested a series of therapeutic nanoparticles for specific targeting of ACPA in synovial fluid.Methods:Nanoparticles were prepared by the microdroplet method and then decorated with synthetic cyclic citrullinated peptide aptamer PEP2, PEG/hexanoic acid and fluorophore (Cy5.5). Nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM) and high-performance liquid chromatography (HPLC). Nanoparticles were then used in a series of in vitro assays, including cell uptake with flow cytometry (FACS) detection, and in vivo studies including disease activity scores, cytokine measurements and near-infrared imaging.Results:We screened a series of citrullinated peptide epitopes and identified a fibrinogen-derived 21-amino-acid-long citrullinated peptide showing high selectivity toward autoantibodies in RA samples. We incorporated this aptamer in the chitosan-hyaluronic acid nanoparticle formulation previously described. Average nanoparticle size was 230 nm ± 10 nm by DLS and SEM; z potential was -0.0012. Purity by HPLC was over 95%. Attachment efficiency of the aptamer was 92% by HPLC. FACS study showed selective uptake of Cy5.5 labelled aptamer-nanoparticle conjugates by neutrophils in the concentration range 0.5-4 nM. Similar to previous studies,1there was no apparent immunogenicity for this nanoparticle formulation measured by cytokine secretion from human peripheral blood leukocytes. In vivo, over 50% reduction of disease activity was achieved in three weeks treatment using as little as 1 nM drug candidate (dosed every 48 hours) in the collagen-induced (CIA) mouse model of RA (N=30; p<0.001 for treated vs placebo). Same was observed in the serum transfer model (N=10). The aptamer-nanoparticle conjugate significantly reduced IL-6 and TNFα levels in the mouse sera (p<0.01). The effects were not inferior to tocilizumab treated controls (N=30). To confirm mode of action, we applied Cy5.5-labelled aptamer-nanoparticles in the collagen-induced mouse model (N=10) and analyzed the resulting uptake by near-infrared imaging. We confirmed over 6-fold higher signal accumulation in inflamed vs healthy joints (p<0.01), which strongly supports the fact that the aptamer is highly specific to the inflammatory process.Conclusion:Overall, we have designed a first-in-class therapeutic nanoparticle drug for specific targeting of anti-citrullinated protein antibodies. The marked effect of this nanoparticle observed in vivo holds promise for targeting ACPAs as a therapeutic option in RA.References:[1]Khatri S, Hansen J, Mendes AC, Chronakis IS, Hung S-C, Mellins ED, Astakhova K. Bioconjug Chem. 2019 Oct 16;30(10):2584–259Disclosure of Interests:Sangita Khatri: None declared, Jonas Hansen: None declared, Mads Hartvig Clausen Shareholder of: iBio Tech ApS, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB., Shu-Chen Hung: None declared, Elisabeth Mellins: None declared, Kira Astakhova: None declared

OP0099 FUNCTIONAL MAPPING OF SYNOVIAL FIBROBLAST POPULATIONS IN HEALTH AND ARTHRITIC DISEASE: INSIGHTS INTO THE PATHOGENIC REMODELING OF SYNOVIAL MICROENVIRONMENT

Background:Our previous studies highlighted the fundamental in vivo role of synovial fibroblasts (SFs) in TNF-mediated murine chronic arthritis1,2and recent findings identified different SF identities based on their transcriptomic profiles with distinct contributions in acute, autoimmunity-based, murine arthritis3.Objectives:In this study, we focus on delineating the map of SF subpopulations in healthy joint and in the course of arthritic disease and the underlying regulatory networks functioning towards pathogenicity.Methods:Sorted single cell suspensions (CD45-, Pdpl+) and their fragmented nuclei from synovial joints of WT, early and late arthritic hTNFtg mice were processed for scRNAseq and scATAC employing a droplet-based technology (10x Genomics). To define the transcriptional and epigenetic signatures originating from the two different assays, we developed an integrative analysis pipeline based on the Seurat software package (v3.1). Meta-analysis of previously reported data of K/BxN serum transfer of arthritis was employed to define commonalities and differences in SF subsets among murine modelled disease.Results:The transition from healthy to chronically affected synovial microenvironment (SME) due to overexpression of hTNF is characterized by a dynamic transformation of SF clusters. The Lining arthritic Thy1lowsynovial layer (L-SFs) is hyper-populated while Sub-Lining Thy1highSF clusters (SL-SFs) are remodeled towards catabolic and inflammatory phenotypes compared to naïve SF organization pattern. Interestingly, trajectory analysis revealed that the SL clusters, which normally exhibit a gradual developmental-like process towards different profiles, differentially change during disease. We identified that the previously reported proliferating SL cluster is absent in healthy synovium, dominates mainly in early stages of chronic arthritis and it is closely related to the L-SFs. Mapping of the gene regulatory networks by RNAseq was supported by scATAC analysis. Similarly, meta-analysis of SF profiles derived from naïve and the K/BxN-serum–treated mice showed significant differences, possibly reflecting the phenotypes of the two established models of arthritis.Conclusion:Our approach unravels for the first time the regulatory heterogeneity and gene expression profiling of SF subpopulations in normal synovium, and reveals deep biological insights of the functional re-organization of SME during development of disease. It further identifies the common and divergent features of the different subtypes of murine arthritis that may well reflect the diversity of RA subtypes and the response to therapies.References:[1]Armaka et al; J Exp Med (2008): 205(2):331-7.[2]Armaka et al; Nat Commun (2018): 9(1):618.[3]Croft et al; Nature (2019): 570(7760):246-251.Disclosure of Interests:None declared

AB0098 THE EFFECT OF MALT1-DEFICIENCY ON THE EFFECTOR PHASE OF EXPERIMENTAL AUTOIMMUNE ARTHRITIS

Background:The paracaspase Malt1 is a cysteine protease, which forms a complex leading to the activation of the in proinflammatory transcription factor NF-κB in lymphocytes with CARMA1 and Bcl10. Previously, we showed that the myeloid equivalent of CARMA1, Card9 is important in neutrophils in Fcγ receptor-mediated cytokine release together with Bcl10 and Malt1. In line with these findings, we observed a significant decrease in the severity of autoantibody-triggered arthritis in the absence of Card9 and Bcl10.Objectives:Our aim was to directly investigate whether the genetic deficiency of Malt1, the third component of the complex altered the process of the K/BxN serum transfer arthritis (that resembles to the effector phase of rheumatoid arthritis).Methods:We used wild type and Malt1–/–mice for our experiments. Autoantibody-mediated arthritis was induced by a single intraperitoneal injection of K/BxN serum. Clinical signs of joint inflammation were scored on a scale based on the cardinal inflammatory clues for two weeks. Ankle thickness was measured by a spring-loaded caliper.Results:Similar to the deficiency of the other two components of the complex, Malt1–/–mice showed a partial, but significant decrease in the macroscopic joint inflammation compared to arthritic serum-treated wild type animals during the entire experimental process. In line with this phenomenon, Malt1–/–animals had reduced autoantibody-triggered ankle thickening.Conclusion:Our results show that Malt1 seems to be an important molecule in the development and progression of experimental autoantibody-induced arthritis in mice, highlighting the role of the molecule as a potential therapeutic target in the future.Disclosure of Interests:None declared