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Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity. The host immune response plays a critical role in the immunopathology of SARS-CoV2. Here the authors combine a systems biology approach to implicate monocytes as key drivers of cytokine storm and disturbed neutrophil activation in COVID-19 disease severity.

Objectives:Patients with rheumatoid arthritis (RA) have defective CD4+CD25+ regulatory T (Treg) cells and increased osteoclastogenesis. A similar situation has been described in collagen-induced arthritis (CIA). In this study, it was investigated whether a single transfer of polyclonally activated Treg cells inhibits CIA and osteoclastogenesis.Methods:Purified Treg cells were expanded in vitro with anti-CD3 and anti-CD28 antibody-coated beads and injected into DBA/1 mice. Mice were immunised with collagen type II (CII) in complete Freund adjuvant (CFA) and scores of arthritis were recorded. In vitro osteoclastogenesis assays were performed on splenocytes by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)κB ligand (RANKL). Levels of anti-CII antibody and cytokines were determined in the supernatant using ELISA and Bio-Plex protein array system.Results:It was found that 106 activated Treg cells significantly counteracted the development of CIA, which was accompanied by decreased serum levels of TNFα and IL6, but not by inhibition of autoimmune antibody responses. The differentiation of osteoclasts in splenocyte cultures was significantly reduced in the presence of prestimulated Treg cells. Expression of cytokines that are described to inhibit osteoclastogenesis, including granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)γ, interleukin (IL)5 and IL10, were dramatically increased upon addition of Treg cells. Furthermore, splenocytes from mice that had been treated with Treg cells displayed an impaired capacity to develop into mature osteoclasts, suggesting that Treg cells abrogated osteoclastogenesis in vivo.Conclusions:Activated CD4+CD25+ Treg cells improve clinical symptoms of CIA, regulate cytokine production and inhibit osteoclastogenesis in vitro and in vivo.

The COVID-19 pandemic poses a major burden on healthcare and economic systems across the globe. Even though a majority of the population develops only minor symptoms upon SARS-CoV-2 infection, a significant number are hospitalized at intensive care units (ICU) requiring critical care. While insights into the early stages of the disease are rapidly expanding, the dynamic immunological processes occurring in critically ill patients throughout their recovery at ICU are far less understood. Here, we have analysed whole blood samples serially collected from 40 surviving COVID-19 patients throughout their recovery in ICU using high-dimensional cytometry by time-of-flight (CyTOF) and cytokine multiplexing. Based on the neutrophil-to-lymphocyte ratio (NLR), we defined four sequential immunotypes during recovery that correlated to various clinical parameters, including the level of respiratory support at concomitant sampling times. We identified classical monocytes as the first immune cell type to recover by restoration of HLA-DR-positivity and the reduction of immunosuppressive CD163 + monocytes, followed by the recovery of CD8 + and CD4 + T cell and non-classical monocyte populations. The identified immunotypes also correlated to aberrant cytokine and acute-phase reactant levels. Finally, integrative analysis of cytokines and immune cell profiles showed a shift from an initially dysregulated immune response to a more coordinated immunogenic interplay, highlighting the importance of longitudinal sampling to understand the pathophysiology underlying recovery from severe COVID-19.

Objectives: To further characterise spontaneous arthritis in aging male DBA/1 mice as a model of spondyloarthropathy and psoriatic arthritis with particular attention to signs of inflammation and nail involvement. Materials and methods: Aging male DBA/1 mice from different litters were caged together (4–6 mice per cage) at the age of 12 weeks, checked twice a week for signs of arthritis, and killed at different times. Hind paws were dissected and processed for histology. Results: Disease incidence varied between 50% and 100% in four different experiments. Besides clinical signs of arthritis, nail abnormalities were noticed. Pathological examination showed the occurrence of dactylitis characterised by diffuse neutrophil infiltration in 6 of 50 paws examined. Onycho-periostitis with progressive destruction of the nail bed and the underlying distal phalanx was seen in 5 of 50 paws examined. Conclusions: Although dactylitis and onychoperiostitis are rare manifestations of the disease process, these data strongly suggest that spontaneous arthritis in aging male DBA/1 mice shares important features with human psoriatic arthritis. This model may therefore be an important tool to study links between stress, sex, inflammation, and new bone formation with particular relevance to human psoriatic arthritis.

Purpose: To verify the antibacterial and immunomodulatory effects of the amylose derivative -- chlorite-oxidized oxyamylose (COAM) -- in a skin wound setting. Methods: In vitro antibacterial effects of COAM against opportunistic bacterial pathogens common to skin wounds, including Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), were determined by cultivation methods. The effects of COAM on myeloid cell infiltration into full thickness skin wounds were investigated in wild-type and in transgenic CX3CR1-GFP mice. Results: On the basis of in vitro experiments, an antibacterial effect of COAM against Staphylococcus species including MRSA was confirmed. The minimum inhibitory concentration of COAM was determined as 2000 [mu]g/mL against these bacterial strains. Control full thickness skin wounds yielded maximal neutrophil influxes and no additive effect on neutrophil influx was observed following topical COAM-treatment. However, COAM administration increased local CX3CR1 macrophage counts at days 3 and 4 and induced a trend towards better wound healing. Conclusion: Aside from its known broad antiviral impact, COAM possesses in vitro antibacterial effects specifically against Gram-positive opportunistic pathogens of the skin and modulates in vivo macrophage contents in mouse skin wounds. Keywords: antimicrobial, wound healing, neutrophils, macrophages, amylose derivative, Staphylococcus

Background Systemic juvenile idiopathic arthritis (sJIA) is one of the most severe pediatric systemic immune-inflammatory disorders, characterized by arthritis and systemic features including fever, rash, lymphadenopathy and leukocytosis. A frequent, potentially fatal complication of sJIA is macrophage activation syndrome (MAS). An excessive production of pro-inflammatory cytokines has been demonstrated in sJIA with or without MAS, yet the exact cause and pathogenesis are still unknown. Studies on gene expression profiles performed on freshly isolated peripheral blood mononuclear cells (PBMCs) from sJIA patients revealed a conspicuous absence of interferon-gamma (IFN-γ)-upregulated genes. This is a peculiar finding, given the highly inflammatory nature of sJIA and the concept that IFN-γ is a driver of MAS. Objectives In this study we explored whether PBMCs of sJIA patients have a defect in IFN-γ signaling by studying induced genes and proteins upon in vitro stimulation with IFN-γ. Methods 9 sJIA patients (7 under treatment and in remission, 1 with active disease despite treatment, 1 untreated) and 1 sJIA patient presenting with MAS were recruited from the University Hospital of Leuven after giving informed consent. PBMCs from patients and healthy controls were obtained by gradient centrifugation and were cultured for 24 hours in the presence or absence of IFN-γ. RNA was extracted from the harvested cells. cDNA was synthesized and qPCR was performed on different IFN-γ-induced genes. IFN-γ-induced proteins (interferon-induced protein (IP-10) and monocyte chemotactic factor-1 (MCP-1)) were analyzed in supernatant of cells, by ELISA. Results In PBMCs from healthy control patients, IFN-γ induced - as expected - upregulation of STAT 1 mRNA, an important IFN-γ-induced transcription factor, as well as other IFN-γ-induced genes such as indoleamine 2,3-dioxygenase (IDO) and IP-10. Some genes such as suppressor of cytokine signaling 3 (SOCS3), MCP-1 and interferon regulatory factor-1 (IRF-1) were not upregulated in healthy PBMCs. Most importantly, all of the IFN γ-associated genes that were significantly induced in healthy control PBMCs were at the least equally well induced in sJIA patients (in both treated, untreated and MAS conditions). Significant production of IP-10 protein was found in the supernatant of IFN-γ-stimulated PBMCs, and here again there was no difference between patients and controls. Conclusions We conclude that PBMCs from sJIA/MAS patients have no intrinsic defect in the IFN-γ machinery. Thus the absence of an IFN-γ signature in sJIA, as reported in gene expression studies, cannot be explained by decreased in vitro responsiveness of PBMCs to IFN-γ, and further research is required to explain this discrepancy. Disclosure of Interest None Declared

Background Systemic juvenile idiopathic arthritis (sJIA) is a disease characterized by arthritis and systemic features such as fever, rash, lymphadenopathy and leukocytosis. The underlying pathogenic mechanism of sJIA is not understood but prolonged stimulation of immune cells and excessive production of cytokines are considered to be important in the pathogenesis of the disease. Objectives The lack of a suitable animal model is a major drawback in the investigation of the pathogenesis of sJIA. We examined whether complete Freund’s adjuvant (CFA), a widely used reagent for chronic stimulation of the immune system, can elicit sJIA-like features in mice. Since interferon-gamma (IFN-γ) exerts key functions in activating and regulating innate and adaptive immune responses and given the disputable role of IFN-γ in sJIA patients, we examined our hypothesis in wild type as well as IFN-γ-deficient mice. Methods IFN-γ-deficient (IFN-γ KO) and wild type mice were injected s.c. with CFA. Body weight, clinical signs of arthritis, complete blood cell counts and peripheral blood biochemistry were monitored. Inflammatory cytokine levels were measured by qPCR and ELISA. Flow cytometry and histology were performed on spleen, liver, bone marrow and lymph nodes. Results Consistent with the immune stimulating properties of CFA, both IFN-γ KO and wild type mice showed splenomegaly and lymphadenopathy from 10 days after CFA challenge onward. Both substrains also developed weight loss; however, while this was transient in wild type animals, it was prolonged and more severe in IFN-γ KO mice. Furthermore, a proportion of the IFN-γ KO, but not wild type mice developed skin rashes as well as redness and swelling in the joints, histologically consistent with prominent synovitis. Complete blood counts demonstrated an increase in platelets and neutrophils in both substrains of mice, the increase being more pronounced in IFN-γ KO mice. IL-6 serum levels were significantly elevated in CFA-challenged IFN-γ KO mice only. Interestingly, between 11 to 40 days post CFA injection, approximately 30% of IFN-γ KO mice developed cytopenia and anemia, as evident from their significantly decreased lymphocyte and red blood cell counts, and decreases in hemoglobin and hematocrit levels. Intriguingly, in CFA-challenged IFN-γ KO mice, bone marrow, spleen, liver and blood showed a prominent infiltration of macrophages, including increased numbers of hemophagocytic macrophages. Conclusions Upon challenge with CFA, IFN-γ KO mice developed clinical, biological and pathological features similar to those seen in patients with sJIA. A proportion of these mice also developed severe inflammation with wasting, anemia and hemophagocytosis, features that are seen in a subset of sJIA patients, diagnosed with macrophage activation syndrome (MAS). To our knowledge, this is the first animal model of sJIA showing an association with MAS. Our data demonstrate that IFN-γ is not required for chronic anemia and hemophagocytosis and challenge the concept that IFN-γ is the critical driver of MAS. Disclosure of Interest None Declared

Rheumatoid arthritis (RA), an autoimmune disease causing inflammation, destruction, and deformity of the joints, affects around 1% of the world population. It is a systemic disease as patients exhibit extra-articular manifestations as well. Collagen-induced arthritis (CIA) in DBA/1 mice is one of the many animal models used to study possible pathogenic mechanisms of RA. It involves immunizing mice with collagen type II in complete Freund's adjuvant. Here we briefly review the general characteristics of RA and CIA and present an overview of data obtained by studying CIA in several gene knockout mice. In particular, detailed analysis of CIA in interferon-gamma (IFN-γ) receptor-deficient mice has pin-pointed IFN-γ as an important cytokine in the pathogenesis and has exposed new functions of IFN-γ in immunological processes. Pilot trials with exogenous IFN-γ in RA have been indicative of a beneficial effect. That improvement of the disease symptoms by IFN-γ treatment was not spectacular may be explained by the fact that RA is a heterogeneous disease in which the severity of the autoimmune disease is strongly determined by environmental factors.

A 49-year-old woman presented with dyspepsia and nocturl regurgitation. A laparoscopic adjustable gastric binding (LAGB) had been performed 6 years before presentation. An upper gastrointestil barium contrast study was performed and revealed a marked dilatation and tortuous course of the esophagus as well as absence of peristalsis and delayed evacuation of the esophagus (Fig. A, B). The findings were compatible with an achalasia-like disorder. An esophageal manometry revealed a constant high LES pressure with aperistalsis, thus confirming the diagnosis of (pseudo-)achalasia. Consequently a complete band deflation was conducted and resulted in a complete resolution of the patient’s symptoms. Two weeks later the control contrast study showed a marked improvement of the delayed evacuation and a small regain of peristaltic function. The dilatation and “sigmoidlike” image of the esophagus remained unchanged (Fig. C).

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease that occurs in 0.5-1.0% of the population worldwide. The primary affected organ is the small diarthrodial joint, where the synovial membrane, cartilage and bone tissue will be damaged, ultimately leading to joint deformity and disability of the patient. In the pathogenesis of RA, the synovial membrane becomes hyperplastic and will be infiltrated with T cells, B cells, neutrophils and macrophages. A hallmark of RA is the progressive destruction of bone tissue caused by an elevated bone resorption by osteoclasts, multinuclear cells derived from the monocyte/macrophage lineage. Objectives Our goal was to provide a method to visualize and quantify joint inflammation by the use of an animal model of RA, namely collagen-induced arthritis (CIA). We focused on the macrophage mannose receptor (MMR), since this protein is a well described marker for macrophages, which are numerously present in inflamed tissues. Methods CIA was induced in DBA/1 mice by the injection of collagen type II in Complete Freund’s adjuvant. Flow cytometry and qPCR were used to study the expression of MMR in vitro in macrophages and osteoclasts and in vivo in CIA. SPECT/CT imaging with 99mTc-labeled nanobodies generated against MMR was performed to visualize and quantify MMR expression in the joints of mice. Results MMR expression was shown to be highly upregulated in cultures of bone marrow-derived macrophages and osteoclasts by qPCR and by flow cytometry using MMR-targeting nanobodies. Ex vivo, we identified MMR in lymph nodes, spleen and bone marrow of naïve and arthritic mice. Interestingly, we detected expression of MMR in the synovial fluid, and to a lesser extent in synovium, of mice with CIA. More specifically, MMR was present on CD11b+F4/80+ macrophages isolated from the synovial fluid of the inflamed joints. SPECT/CT imaging was used to detect MMR in vivo in mice with CIA. Therefore, nanobodies against MMR were radioactively labeled with 99mTc, while nanobodies targeting a bacterial enzyme were used as controls. We observed high signals of MMR in lymph nodes, spleen and liver in naïve conditions as well as after immunization. Importantly, the joints of arthritic mice displayed high retention of MMR nanobody. The signal from SPECT imaging was significantly higher in mice with arthritic symptoms compared to naïve animals or immunized mice without clinical symptoms. Conclusions The use of MMR nanobodies in SPECT/CT imaging generates the possibility to track and quantify inflammatory macrophages in vivo in arthritic joints. In vivo quantification of joint inflammation by non-invasive techniques would be a great help in diagnosis and monitoring of disease processes as well as testing the efficiency of (new) drugs. Disclosure of Interest None Declared