Murine Models of Secondary Cytokine Storm Syndromes.

Hemophagocytic lymphohistiocytosis (HLH) comprises a broad spectrum of life-threatening cytokine storm syndromes, classified into primary (genetic) or secondary (acquired) HLH. The latter occurs in a variety of medical conditions, including infections, malignancies, autoimmune and autoinflammatory diseases, acquired immunodeficiency, and metabolic disorders. Despite recent advances in the field, the pathogenesis of secondary HLH remains incompletely understood. Considering the heterogeneity of triggering factors and underlying diseases in secondary HLH, a large diversity of animal models has been developed to explore pivotal disease mechanisms. To date, over 20 animal models have been described that each recapitulates certain aspects of secondary HLH. This review provides a comprehensive overview of the existing models, highlighting relevant findings, discussing the involvement of different cell types and cytokines in disease development and progression, and considering points of interest toward future therapeutic strategies.

Effect on neutrophil migration and antimicrobial functions by the Bruton's tyrosine kinase inhibitors tolebrutinib, evobrutinib and fenebrutinib.

Multiple sclerosis (MS) is a neurodegenerative, autoimmune disease that is still incurable. Nowadays, a variety of new drugs are being developed to prevent excessive inflammation and halt neurodegeneration. Among these are the inhibitors of Bruton's tyrosine kinase (BTK). Being indispensable for B cells, this enzyme became an appealing therapeutic target for autoimmune diseases. Recognizing the emerging importance of BTK in myeloid cells, we investigated the impact of upcoming BTK inhibitors on neutrophil functions. Although adaptive immunity in MS has been thoroughly studied, unanswered questions about the pathogenesis can be addressed by studying the effects of candidate MS drugs on innate immune cells such as neutrophils, previously overlooked in MS. In this study, we used three BTK inhibitors (evobrutinib, fenebrutinib and tolebrutinib), and found that they reduce neutrophil activation by the bacterial peptide N-formylmethionyl-leucyl-phenylalanine and the chemokine interleukin 8/CXCL8. Furthermore, they diminished the production of reactive oxygen species and release of neutrophil extracellular traps. Additionally, the production of CXCL8 and interleukin-1ß in response to inflammatory stimuli was decreased. Inhibitory effects of the drugs on neutrophil activation were not related to toxicity. Instead, BTK inhibitors prolonged neutrophil survival in an inflammatory environment. Finally, treatment with BTK inhibitors decreased neutrophil migration towards CXCL8 in a Boyden chamber assay but not in a trans endothelial set-up. Also, in vivo CXCL1-induced migration was unaffected by BTK inhibitors. Collectively, this study provides novel insights into the impact of BTK inhibitors on neutrophil functions, thereby holding important implications for autoimmune or hematological diseases where BTK is crucial.

CHIT1 at diagnosis predicts faster disability progression and reflects early microglial activation in multiple sclerosis.

Multiple sclerosis (MS) is characterized by heterogeneity in disease course and prediction of long-term outcome remains a major challenge. Here, we investigate five myeloid markers - CHIT1, CHI3L1, sTREM2, GPNMB and CCL18 - in the cerebrospinal fluid (CSF) at diagnostic lumbar puncture in a longitudinal cohort of 192 MS patients. Through mixed-effects and machine learning models, we show that CHIT1 is a robust predictor for faster disability progression. Integrative analysis of 11 CSF and 26 central nervous system (CNS) parenchyma single-cell/nucleus RNA sequencing samples reveals CHIT1 to be predominantly expressed by microglia located in active MS lesions and enriched for lipid metabolism pathways. Furthermore, we find CHIT1 expression to accompany the transition from a homeostatic towards a more activated, MS-associated cell state in microglia. Neuropathological evaluation in post-mortem tissue from 12 MS patients confirms CHIT1 production by lipid-laden phagocytes in actively demyelinating lesions, already in early disease stages. Altogether, we provide a rationale for CHIT1 as an early biomarker for faster disability progression in MS.

Aspecific binding of anti-NK1.1 antibodies on myeloid cells in an experimental model for malaria-associated acute respiratory distress syndrome.

BACKGROUND: Conventional natural killer (cNK) cells play an important role in the innate immune response by directly killing infected and malignant cells and by producing pro- and anti-inflammatory cytokines. Studies on their role in malaria and its complications have resulted in conflicting results. METHODS: Using the commonly used anti-NK1.1 depletion antibodies (PK136) in an in-house optimized experimental model for malaria-associated acute respiratory distress syndrome (MA-ARDS), the role of cNK cells was investigated. Moreover, flow cytometry was performed to characterize different NK cell populations. RESULTS: While cNK cells were found to be dispensable in the development of MA-ARDS, the appearance of a NK1.1+ cell population was observed in the lungs upon infection despite depletion with anti-NK1.1. Detailed characterization of the unknown population revealed that this population consisted of a mixture of monocytes and macrophages that bind the anti-NK1.1 antibody in an aspecific way. This aspecific binding may occur via Fcγ receptors, such as FcγR4. In contrast, in vivo depletion using anti-NK1.1 antibodies was proved to be specific for cNK cells. CONCLUSION: cNK cells are dispensable in the development of experimental MA-ARDS. Moreover, careful flow cytometric analysis, with a critical mindset in relation to potential aspecific binding despite the use of commercially available Fc blocking reagents, is critical to avoid misinterpretation of the results.

Biallelic mutations in the CFHR genes underlying atypical hemolytic uremic syndrome in a patient with catastrophic adult-onset Still's disease and recurrent macrophage activation syndrome: A case report.

We report the fatal case of a 20-year-old woman with refractory adult-onset Still's disease (AOSD) accompanied by fulminant macrophage activation syndrome (MAS) and atypical hemolytic uremic syndrome (aHUS). Anakinra and tocilizumab temporarily controlled AOSD. In 2021, she presented to ICU with generalized tonic-clonic seizure, lymphocytic aseptic meningitis, and acute kidney injury. Despite hemodialysis and methylprednisolone, she developed another seizure, MAS, and disseminated intravascular coagulation (DIC). Following brief control, MAS flares -reflected by increased plasma CXCL9 and CXCL10- re-emerged and were controlled through dexamethasone, etoposide, cyclosporin and tofacitinib. No mutations were detected in haemophagocytic lymphohistiocytosis (HLH)-associated genes, nor in genes associated with periodic fever syndromes. Post-mortem genetic testing revealed loss-of-function biallelic deletions in complement factor H-related proteins (CFHR) genes, predisposing aHUS. This case underscores the importance of prompt genetic assessment of complement-encoding alleles, in addition to HLH-related genes, in patients with severe AOSD with recurrent MAS and features of thrombotic microangiopathy (TMA).

Physiological fibrin hydrogel modulates immune cells and molecules and accelerates mouse skin wound healing.

Introduction: Wound healing is a complex process to restore homeostasis after injury and insufficient skin wound healing is a considerable problem in medicine. Whereas many attempts of regenerative medicine have been made for wound healing with growth factors and cell therapies, simple pharmacological and immunological studies are lagging behind. We investigated how fibrin hydrogels modulate immune cells and molecules in skin wound healing in mice. Methods: Physiological fibrin hydrogels (3.5 mg/mL fibrinogen) were generated, biophysically analyzed for stiffness and protein contents and were structurally studied by scanning electron microscopy. Physiological fibrin hydrogels were applied to full thickness skin wounds and, after 3 days, cells and molecules in wound tissues were analyzed. Leukocytes, endothelial cells, fibroblasts and keratinocytes were explored with the use of Flow Cytometry, whereas cytokines and matrix metalloproteinases were analyzed with the use of qPCR, ELISAs and zymography. Skin wound healing was analyzed microscopically at day 3, macroscopically followed daily during repair in mice and compared with commercially available fibrin sealant Tisseel. Results: Exogenous fibrin at physiological concentrations decreased neutrophil and increased non-classical Ly6Clow monocyte and resolutive macrophage (CD206+ and CX3CR1+) populations, at day 3 after injury. Fibrin hydrogel reduced the expression of pro-inflammatory cytokines and increased IL-10 levels. In line with these findings, gelatinase B/MMP-9 was decreased, whereas gelatinase A/MMP-2 levels remained unaltered. Frequencies of dermal endothelial cells, fibroblasts and keratinocytes were increased and keratinocyte migration was enhanced by fibrin hydrogel. Importantly, physiological fibrin accelerated the healing of skin wounds in contrast to the highly concentrated fibrin sealant Tisseel, which delayed wound repair and possessed a higher fiber density. Conclusion: Collectively, we show that adding a tailored fibrin hydrogel scaffold to a wound bed positively influences the healing process, modulating leukocyte populations and inflammatory responses towards a faster wound repair.

Homozygous DBF4 mutation as a cause of severe congenital neutropenia.

BACKGROUND: Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases. OBJECTIVE: We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. METHODS: Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. RESULTS: We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. CONCLUSION: Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.

Novel method to quantify peptidylarginine deiminase activity shows distinct citrullination patterns in rheumatoid and juvenile idiopathic arthritis.

Introduction: Peptidylarginine deiminases (PADs) mediate citrullination, an irreversible posttranslational modification that converts arginine to citrulline residues in proteins. Rheumatoid arthritis (RA) is characterized by unique autoantibodies that recognize citrullinated peptides, which are highly specific for this disease. However, the mechanism preceding the anti-citrulline response remains largely unclear. PAD enzymes are known to fuel the autoimmune response by generating autoreactive epitopes, and sustain local synovial inflammation through neutrophil extracellular trap formation. Therefore, detecting endogenous PAD activity is important to understand the pathogenesis of arthritis. Methods: In this study, we improved a fluorescent in vitro assay to enable endogenous PAD activity characterization in complex samples. We combine the use of an in-house synthetic, arginine-rich substrate and a negatively charged dye molecule to visualize enzyme activity. Results: This pioneering PAD assay allowed profiling of active citrullination in leukocytes and in local and systemic samples of an arthritis cohort. Our results reveal that RA and juvenile idiopathic arthritis (JIA) synovial fluids display similar levels of PAD activity. In contrast, citrullination was limited in joints of patients suffering from gout or Lyme's disease. Interestingly, in blood, a higher level of extracellular citrullination was only found in anti-CCP-positive RA patients. Discussion: Our finding suggests that enhanced synovial PAD activity drives the loss in tolerance towards citrullinated proteins and that systemic citrullination may indicate the risk for developing citrulline-specific autoimmunity.

Multinucleation resets human macrophages for specialized functions at the expense of their identity.

Macrophages undergo plasma membrane fusion and cell multinucleation to form multinucleated giant cells (MGCs) such as osteoclasts in bone, Langhans giant cells (LGCs) as part of granulomas or foreign-body giant cells (FBGCs) in reaction to exogenous material. How multinucleation per se contributes to functional specialization of mature mononuclear macrophages remains poorly understood in humans. Here, we integrate comparative transcriptomics with functional assays in purified mature mononuclear and multinucleated human osteoclasts, LGCs and FBGCs. Strikingly, in all three types of MGCs, multinucleation causes a pronounced downregulation of macrophage identity. We show enhanced lysosome-mediated intracellular iron homeostasis promoting MGC formation. The transition from mononuclear to multinuclear state is accompanied by cell specialization specific to each polykaryon. Enhanced phagocytic and mitochondrial function associate with FBGCs and osteoclasts, respectively. Moreover, human LGCs preferentially express B7-H3 (CD276) and can form granuloma-like clusters in vitro, suggesting that their multinucleation potentiates T cell activation. These findings demonstrate how cell-cell fusion and multinucleation reset human macrophage identity as part of an advanced maturation step that confers MGC-specific functionality.

IRF2 is required for development and functional maturation of human NK cells.

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells.

Severe COVID-19 patients display hyper-activated NK cells and NK cell-platelet aggregates.

COVID-19 is characterised by a broad spectrum of clinical and pathological features. Natural killer (NK) cells play an important role in innate immune responses to viral infections. Here, we analysed the phenotype and activity of NK cells in the blood of COVID-19 patients using flow cytometry, single-cell RNA-sequencing (scRNA-seq), and a cytotoxic killing assay. In the plasma of patients, we quantified the main cytokines and chemokines. Our cohort comprises COVID-19 patients hospitalised in a low-care ward unit (WARD), patients with severe COVID-19 disease symptoms hospitalised in intensive care units (ICU), and post-COVID-19 patients, who were discharged from hospital six weeks earlier. NK cells from hospitalised COVID-19 patients displayed an activated phenotype with substantial differences between WARD and ICU patients and the timing when samples were taken post-onset of symptoms. While NK cells from COVID-19 patients at an early stage of infection showed increased expression of the cytotoxic molecules perforin and granzyme A and B, NK cells from patients at later stages of COVID-19 presented enhanced levels of IFN-γ and TNF-α which were measured ex vivo in the absence of usual in vitro stimulation. These activated NK cells were phenotyped as CD49a+CD69a+CD107a+ cells, and their emergence in patients correlated to the number of neutrophils, and plasma IL-15, a key cytokine in NK cell activation. Despite lower amounts of cytotoxic molecules in NK cells of patients with severe symptoms, majority of COVID-19 patients displayed a normal cytotoxic killing of Raji tumour target cells. In vitro stimulation of patients blood cells by IL-12+IL-18 revealed a defective IFN-γ production in NK cells of ICU patients only, indicative of an exhausted phenotype. ScRNA-seq revealed, predominantly in patients with severe COVID-19 disease symptoms, the emergence of an NK cell subset with a platelet gene signature that we identified by flow and imaging cytometry as aggregates of NK cells with CD42a+CD62P+ activated platelets. Post-COVID-19 patients show slow recovery of NK cell frequencies and phenotype. Our study points to substantial changes in NK cell phenotype during COVID-19 disease and forms a basis to explore the contribution of platelet-NK cell aggregates to antiviral immunity against SARS-CoV-2 and disease pathology.

Chlorite-Oxidized Oxyamylose (COAM) Has Antibacterial Activity and Positively Affects Skin Wound Healing.

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 µg/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.

Live-attenuated YF17D-vectored COVID-19 vaccine protects from lethal yellow fever virus infection in mouse and hamster models.

BACKGROUND: The live-attenuated yellow fever vaccine YF17D holds great promise as alternative viral vector vaccine platform, showcased by our previously presented potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidate YF-S0. Besides protection from SARS-CoV-2, YF-S0 also induced strong yellow fever virus (YFV)-specific immunity, suggestive for full dual activity. A vaccine concomitantly protecting from SARS-CoV-2 and YFV would be of great benefit for those living in YFV-endemic areas with limited access to current SARS-CoV-2 vaccines. However, for broader applicability, pre-existing vector immunity should not impact the potency of such YF17D-vectored vaccines. METHODS: The immunogenicity and efficacy of YF-S0 against YFV and SARS-CoV-2 in the presence of strong pre-existing YFV immunity were evaluated in mouse and hamster challenge models. FINDINGS: Here, we show that a single dose of YF-S0 is sufficient to induce strong humoral and cellular immunity against YFV as well as SARS-CoV-2 in mice and hamsters; resulting in full protection from vigorous YFV challenge in either model; in mice against lethal intracranial YF17D challenge, and in hamsters against viscerotropic infection and liver disease following challenge with highly pathogenic hamster-adapted YFV-Asibi strain. Importantly, strong pre-existing immunity against the YF17D vector did not interfere with subsequent YF-S0 vaccination in mice or hamsters; nor with protection conferred against SARS-CoV-2 strain B1.1.7 (Alpha variant) infection in hamsters. INTERPRETATION: Our findings warrant the development of YF-S0 as dual SARS-CoV-2 and YFV vaccine. Contrary to other viral vaccine platforms, use of YF17D does not suffer from pre-existing vector immunity. FUNDING: Stated in the acknowledgments.

The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency.

Natural killer (NK) cells are innate lymphocytes that eliminate virus-infected and cancer cells by cytotoxicity and cytokine secretion. In addition to circulating NK cells, distinct tissue-resident NK subsets have been identified in various organs. Although transcription factors regulating NK cell development and function have been extensively studied in mice, the role of RUNX2 in these processes has not been investigated, neither in mice nor in human. Here, by manipulating RUNX2 expression with either knockdown or overexpression in human haematopoietic stem cell-based NK cell differentiation cultures, combined with transcriptomic and ChIP-sequencing analyses, we established that RUNX2 drives the generation of NK cells, possibly through induction of IL-2Rß expression in NK progenitor cells. Importantly, RUNX2 promotes tissue residency in human NK cells. Our findings have the potential to improve existing NK cell-based cancer therapies and can impact research fields beyond NK cell biology, since tissue-resident subsets have also been described in other lymphocyte subpopulations.

Natural Killer Cells in Multiple Sclerosis: Entering the Stage.

Studies investigating the immunopathology of multiple sclerosis (MS) have largely focused on adaptive T and B lymphocytes. However, in recent years there has been an increased interest in the contribution of innate immune cells, amongst which the natural killer (NK) cells. Apart from their canonical role of controlling viral infections, cell stress and malignancies, NK cells are increasingly being recognized for their modulating effect on the adaptive immune system, both in health and autoimmune disease. From different lines of research there is now evidence that NK cells contribute to MS immunopathology. In this review, we provide an overview of studies that have investigated the role of NK cells in the pathogenesis of MS by use of the experimental autoimmune encephalomyelitis (EAE) animal model, MS genetics or through ex vivo and in vitro work into the immunology of MS patients. With the advent of modern hypothesis-free technologies such as single-cell transcriptomics, we are exposing an unexpected NK cell heterogeneity, increasingly blurring the boundaries between adaptive and innate immunity. We conclude that unravelling this heterogeneity, as well as the mechanistic link between innate and adaptive immune cell functions will lay the foundation for the use of NK cells as prognostic tools and therapeutic targets in MS and a myriad of other currently uncurable autoimmune disorders.

Multinucleated Giant Cells: Current Insights in Phenotype, Biological Activities, and Mechanism of Formation.

Monocytes and macrophages are innate immune cells with diverse functions ranging from phagocytosis of microorganisms to forming a bridge with the adaptive immune system. A lesser-known attribute of macrophages is their ability to fuse with each other to form multinucleated giant cells. Based on their morphology and functional characteristics, there are in general three types of multinucleated giant cells including osteoclasts, foreign body giant cells and Langhans giant cells. Osteoclasts are bone resorbing cells and under physiological conditions they participate in bone remodeling. However, under pathological conditions such as rheumatoid arthritis and osteoporosis, osteoclasts are responsible for bone destruction and bone loss. Foreign body giant cells and Langhans giant cells appear only under pathological conditions. While foreign body giant cells are found in immune reactions against foreign material, including implants, Langhans giant cells are associated with granulomas in infectious and non-infectious diseases. The functionality and fusion mechanism of osteoclasts are being elucidated, however, our knowledge on the functions of foreign body giant cells and Langhans giant cells is limited. In this review, we describe and compare the phenotypic aspects, biological and functional activities of the three types of multinucleated giant cells. Furthermore, we provide an overview of the multinucleation process and highlight key molecules in the different phases of macrophage fusion.

Role for Granulocyte Colony-Stimulating Factor in Neutrophilic Extramedullary Myelopoiesis in a Murine Model of Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: Systemic juvenile idiopathic arthritis (JIA) is a systemic inflammatory disease with childhood onset. Systemic JIA is associated with neutrophilia, including immature granulocytes, potentially driven by the growth factor granulocyte-colony stimulating factor (G-CSF). This study was undertaken to investigate the role of G-CSF in the pathology of systemic JIA. METHODS: Injection of Freund's complete adjuvant (CFA) in BALB/c mice induces mild inflammation and neutrophilia in wild-type (WT) mice and a more pronounced disease, reminiscent to that of JIA patients, in interferon-γ-knockout (IFNγ-KO) mice. Extramedullary myelopoiesis was studied in CFA-immunized mice by single-cell RNA sequencing, and the effect of G-CSF receptor (G-CSFR) blockage on neutrophil development and systemic JIA pathology was evaluated. Additionally, plasma G-CSF levels were measured in patients. RESULTS: Both in systemic JIA patients and in a corresponding mouse model, plasma G-CSF levels were increased. In the mouse model, we demonstrated that G-CSF is responsible for the observed neutrophilia and extramedullary myelopoiesis and the induction of immature neutrophils and myeloid-derived suppressor-like cells. Administration of a G-CSFR antagonizing antibody blocked the maturation and differentiation of neutrophils in CFA-immunized mice. In IFNγ-KO mice, treatment was associated with almost complete inhibition of arthritis due to reduced neutrophilia and osteoclast formation. Disease symptoms were ameliorated, but slight increases in interleukin-6 (IL-6), tumor necrosis factor, and IL-17 were detected upon G-CSFR inhibition in the IFNγ-KO mice, and were associated with mild increases in weight loss, tail damage, and immature red blood cells. CONCLUSION: We describe the role of G-CSF in a mouse model of systemic JIA and suggest an important role for G-CSF-induced myelopoiesis and neutrophilia in regulating the development of arthritis.