Dupilumab-induced eosinophilia in patients with diffuse type 2 chronic rhinosinusitis.

BACKGROUND Dupilumab, a monoclonal anti-IL-4Rα antibody, is approved for several type 2 mediated inflammatory diseases like asthma, atopic dermatitis, and diffuse type 2 chronic rhinosinusitis (CRS). Clinical studies had reported a transient increase in blood eosinophils during dupilumab therapy. This study aimed to assess the impact of elevated blood eosinophils on clinical outcome and to investigate the cause of high blood eosinophil levels under dupilumab therapy. METHODS Patients suffering from diffuse type 2 CRS treated with dupilumab were examined on days 0, 28, 90, and 180 after therapy start. Sino-Nasal-Outcome-Test Score (SNOT-22), Total Nasal Polyp Score (TNPS), and blood samples were collected. Cytokine measurements and proteomics analysis were conducted. Flow cytometry analysis measured receptor expression on eosinophils. RESULTS Sixty-eighty patients were included. Baseline eosinophilia ≥0.3G/L was observed in 63.2% of patients, and in 30.9% of patients, eosinophils increased by ≥0.5G/L under dupilumab. Subjects with eosinophilia ≥0.3G/L at baseline had the best SNOT-22 mean change compared to no eosinophilia. Eosinophil elevation during dupilumab therapy had no impact on clinical scores. The eosinophil adhesion molecule VCAM-1 decreased significantly during therapy in all patients. The chemokine receptor CXCR4 was significantly down- and IL-4 upregulated in subjects with eosinophil increase. CONCLUSION Our findings suggest that increased eosinophils in type 2 CRS are associated with a good clinical response to dupilumab. Patients with elevated IL-4 at baseline developed dupilumab-induced transient eosinophilia. We identified the downregulation of VCAM-1 and surface markers CD49d and CXCR4 on eosinophils as possible explanations of dupilumab-induced eosinophilia

Assessment of treatment response in cardiac sarcoidosis based on myocardial 18^{18}F-FDG uptake

OBJECTIVE Immunosuppressive therapy for cardiac sarcoidosis (CS) still largely consists of corticosteroid monotherapy. However, high relapse rates after tapering and insufficient efficacy are significant problems. The objective of this study was to investigate the efficacy and safety of non-biological and biological disease-modifying anti-rheumatic drugs (nb/bDMARDs) considering control of myocardial inflammation assessed by $^{18}$F-fluorodeoxyglucose positron emission tomography/computed tomography ($^{18}$F-FDG PET/CT) of the heart. METHODS We conducted a retrospective analysis of treatment response to nb/bDMARDs of all CS patients seen in the sarcoidosis center of the University Hospital Zurich between January 2016 and December 2020. RESULTS We identified 50 patients with CS. Forty-five patients with at least one follow-up PET/CT scan were followed up for a mean of 20.5 ± 12.8 months. Most of the patients were treated with prednisone and concomitant nb/bDMARDs. At the first follow-up PET/CT scan after approximately 6.7 ± 3 months, only adalimumab showed a significant reduction in cardiac metabolic activity. Furthermore, comparing all serial follow-up PET/CT scans (143), tumor necrosis factor inhibitor (TNFi)-based therapies showed statistically significant better suppression of myocardial $^{18}$F-FDG uptake compared to other treatment regimens. On the last follow-up, most adalimumab-treated patients were inactive (n = 15, 48%) or remitting (n = 11, 35%), and only five patients (16%) were progressive. TNFi was safe even in patients with severely reduced left ventricular ejection fraction (LVEF), and a significant improvement in LVEF under TNFi treatment was observed. CONCLUSION TNFi shows better control of myocardial inflammation compared to nbDMARDs and corticosteroid monotherapies in patients with CS. TNFi was efficient and safe even in patients with severely reduced LVEF

Assessment of treatment response in cardiac sarcoidosis based on myocardial 18F-FDG uptake

ObjectiveImmunosuppressive therapy for cardiac sarcoidosis (CS) still largely consists of corticosteroid monotherapy. However, high relapse rates after tapering and insufficient efficacy are significant problems. The objective of this study was to investigate the efficacy and safety of non-biological and biological disease-modifying anti-rheumatic drugs (nb/bDMARDs) considering control of myocardial inflammation assessed by 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) of the heart.MethodsWe conducted a retrospective analysis of treatment response to nb/bDMARDs of all CS patients seen in the sarcoidosis center of the University Hospital Zurich between January 2016 and December 2020.ResultsWe identified 50 patients with CS. Forty-five patients with at least one follow-up PET/CT scan were followed up for a mean of 20.5 ± 12.8 months. Most of the patients were treated with prednisone and concomitant nb/bDMARDs. At the first follow-up PET/CT scan after approximately 6.7 ± 3 months, only adalimumab showed a significant reduction in cardiac metabolic activity. Furthermore, comparing all serial follow-up PET/CT scans (143), tumor necrosis factor inhibitor (TNFi)-based therapies showed statistically significant better suppression of myocardial 18F-FDG uptake compared to other treatment regimens. On the last follow-up, most adalimumab-treated patients were inactive (n = 15, 48%) or remitting (n = 11, 35%), and only five patients (16%) were progressive. TNFi was safe even in patients with severely reduced left ventricular ejection fraction (LVEF), and a significant improvement in LVEF under TNFi treatment was observed.ConclusionTNFi shows better control of myocardial inflammation compared to nbDMARDs and corticosteroid monotherapies in patients with CS. TNFi was efficient and safe even in patients with severely reduced LVEF

Iron accelerates hemoglobin oxidation increasing mortality in vascular diseased guinea pigs following transfusion of stored blood

Non-transferrin-bound iron (NTBI) and free hemoglobin (Hb) accumulate in circulation following stored RBC transfusions. This study investigated transfusion, vascular disease, and mortality in guinea pigs after stored RBC transfusion alone and following cotransfusion with apo-transferrin (apo-Tf) and haptoglobin (Hp). The effects of RBC exchange transfusion dose (1, 3, and 9 units), storage period (14 days), and mortality were evaluated in guinea pigs with a vascular disease phenotype. Seven-day mortality and the interaction between iron and Hb as cocontributors to adverse outcome were studied. Concentrations of iron and free Hb were greatest after transfusion with 9 units of stored RBCs compared with fresh RBCs or stored RBCs at 1- and 3-unit volumes. Nine units of stored RBCs led to mortality in vascular diseased animals, but not normal animals. One and 3 units of stored RBCs did not cause a mortality effect, suggesting the concomitant relevance of NTBI and Hb on outcome. Cotransfusion with apo-Tf or Hp restored survival to 100% following 9-unit RBC transfusions in vascular diseased animals. Our data suggest that increases in plasma NTBI and Hb contribute to vascular disease-associated mortality through iron-enhanced Hb oxidation and enhanced tissue injury

Agonistic Anti-CD40 Antibody Triggers an Acute Liver Crisis With Systemic Inflammation in Humanized Sickle Cell Disease Mice

Sickle cell disease (SCD) is an inherited hemolytic disorder, defined by a point mutation in the β-globin gene. Stress conditions such as infection, inflammation, dehydration, and hypoxia trigger erythrocyte sickling. Sickled red blood cells (RBCs) hemolyze more rapidly, show impaired deformability, and increased adhesive properties to the endothelium. In a proinflammatory, pro-coagulative environment with preexisting endothelial dysfunction, sickled RBCs promote vascular occlusion. Hepatobiliary involvement related to the sickling process, such as an acute sickle hepatic crisis, is observed in about 10% of acute sickle cell crisis incidents. In mice, ligation of CD40 with an agonistic antibody leads to a macrophage activation in the liver, triggering a sequence of systemic inflammation, endothelial cell activation, thrombosis, and focal ischemia. We found that anti-CD40 antibody injection in sickle cell mice induces a systemic inflammatory and hemodynamic response with accelerated hemolysis, extensive vaso-occlusion, and large ischemic infarctions in the liver mimicking an acute hepatic crisis. Administration of the tumor necrosis factor-α (TNF-α) blocker, etanercept, and the heme scavenger protein, hemopexin attenuated end-organ damage. These data collectively suggest that anti-CD40 administration offers a novel acute liver crisis model in humanized sickle mice, allowing for evaluation of therapeutic proof-of-concept

Dupilumab‐induced eosinophilia in patients with diffuse type 2 chronic rhinosinusitis

BackgroundDupilumab, a monoclonal anti‐IL‐4Rα antibody, is approved for several type 2 mediated inflammatory diseases like asthma, atopic dermatitis, and diffuse type 2 chronic rhinosinusitis (CRS). Clinical studies had reported a transient increase in blood eosinophils during dupilumab therapy.This study aimed to assess the impact of elevated blood eosinophils on clinical outcome and to investigate the cause of high blood eosinophil levels under dupilumab therapy.MethodsPatients suffering from diffuse type 2 CRS treated with dupilumab were examined on days 0, 28, 90, and 180 after therapy start. Sino‐Nasal‐Outcome‐Test Score (SNOT‐22), Total Nasal Polyp Score (TNPS), and blood samples were collected. Cytokine measurements and proteomics analysis were conducted. Flow cytometry analysis measured receptor expression on eosinophils.ResultsSixty‐eighty patients were included. Baseline eosinophilia ≥0.3G/L was observed in 63.2% of patients, and in 30.9% of patients, eosinophils increased by ≥0.5G/L under dupilumab. Subjects with eosinophilia ≥0.3G/L at baseline had the best SNOT‐22 mean change compared to no eosinophilia. Eosinophil elevation during dupilumab therapy had no impact on clinical scores. The eosinophil adhesion molecule VCAM‐1 decreased significantly during therapy in all patients. The chemokine receptor CXCR4 was significantly down‐ and IL‐4 upregulated in subjects with eosinophil increase.ConclusionOur findings suggest that increased eosinophils in type 2 CRS are associated with a good clinical response to dupilumab. Patients with elevated IL‐4 at baseline developed dupilumab‐induced transient eosinophilia. We identified the downregulation of VCAM‐1 and surface markers CD49d and CXCR4 on eosinophils as possible explanations of dupilumab‐induced eosinophilia

Hemolysis transforms liver macrophages into anti-inflammatory erythrophagocytes

During hemolysis, macrophages in the liver phagocytose damaged erythrocytes to prevent the toxic effects of cell-free hemoglobin and heme. It remains unclear how this homeostatic process modulates phagocyte functions in inflammatory diseases. Using a genetic mouse model of spherocytosis and single-cell RNA sequencing, we found that erythrophagocytosis skewed liver macrophages into a unique anti-inflammatory phenotype that we defined as Marcohigh/Hmoxhigh/MHC-class IIlow erythrophagocytes. This phenotype transformation profoundly mitigated disease expression in a model of an anti-CD40-induced hyperinflammatory syndrome with necrotic hepatitis and in a non-alcoholic steatohepatitis model, representing two macrophage-driven sterile inflammatory diseases. We reproduced the anti-inflammatory erythrophagocyte transformation in vitro by heme-exposure of mouse and human macrophages, yielding a distinctive transcriptional signature that segregated heme-polarized from M1- and M2-polarized cells. Mapping transposase-accessible chromatin in single cells by sequencing (scATAC-seq) defined the transcription factor NFE2L2/NRF2 as a critical driver of erythrophagocytes, and Nfe2l2/Nrf2-deficiency restored heme-suppressed inflammation. Our findings point to a pathway that regulates macrophage functions to link erythrocyte homeostasis with innate immunity

Phenotype-specific recombinant haptoglobin polymers co-expressed with C1r-like protein as optimized hemoglobin-binding therapeutics

BACKGROUND: Preclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1-1 versus Hp 2-2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA. ProHp is proteolytically cleaved by complement C1r subcomponent-like protein (C1r-LP) in the endoplasmic reticulum. Two main allelic Hp variants, HP1 and HP2 exist. The larger HP2 is considered to be the ancestor variant of all human Hp alleles and is characterized by an α2-chain, which contains an extra cysteine residue that pairs with additional α-chains generating multimers with molecular weights of 200-900 kDa. The two human HP1 alleles (HP1F and HP1S) differ by a two-amino-acid substitution polymorphism within the α-chain and are derived from HP2 by recurring exon deletions. RESULTS: In the present study, we describe a process for the production of recombinant phenotype specific Hp polymers in mammalian FS293F cells. This approach demonstrates that efficient expression of mature and fully functional protein products requires co-expression of active C1r-LP. The functional characterization of our proteins, which included monomer/polymer distribution, binding affinities as well as NO-sparing and antioxidant functions, demonstrated that C1r-LP-processed recombinant Hp demonstrates equal protective functions as plasma derived Hp in vitro as well as in animal studies. CONCLUSIONS: We present a recombinant production process for fully functional phenotype-specific Hp therapeutics. The proposed process could accelerate the development of Hb scavengers to treat patients with cell-free Hb associated disease states, such as sickle cell disease and other hemolytic conditions

Line-selective macrophage activation with an anti-CD40 antibody drives a hemophagocytic syndrome in mice

Hemophagocytic syndromes comprise a cluster of hyperinflammatory disorders, including hemophagocytic lymphohistiocytosis and macrophage activation syndrome. Overwhelming macrophage activation has long been considered a final common pathway in the pathophysiology of hemophagocytic syndromes leading to the characteristic cytokine storm, laboratory abnormalities, and organ injuries that define the clinical spectrum of the disease. So far, it is unknown whether primary macrophage activation alone can induce the disease phenotype. In this study, we established a novel mouse model of a hemophagocytic syndrome by treating mice with an agonistic anti-CD40 antibody (Ab). The response in wild-type mice is characterized by a cytokine storm, associated with hyperferritinemia, high soluble CD25, erythrophagocytosis, secondary endothelial activation with multiple organ vaso-occlusion, necrotizing hepatitis, and variable cytopenias. The disease is dependent on a tumor necrosis factor-α-interferon-γ-driven amplification loop. After macrophage depletion with clodronate liposomes or in mice with a macrophage-selective deletion of the CD40 gene (CD40flox/flox/LysMCre), the disease was abolished. These data provide a new preclinical model of a hemophagocytic syndrome and reinforce the key pathophysiological role of macrophages

Reversal of hemochromatosis by apo-transferrin in non-transfused and transfused Hbbth3/+ (heterozygous b1/ b2 globin gene deletion) mice

Intermediate beta-thalassemia demonstrates a broad spectrum of sequelae and may require occasional blood transfusions over a lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron (NTBI) in plasma. The iron scavenger and transport protein transferrin (Tf) is a potential treatment being studied for correction of anemia. However, Tf may also function to prevent or reduce iron loading of tissues when exposures to NTBI increase. Here we evaluate the effects of apoTf dosing on tissue iron loading and early tissue pathology in non-transfused and transfused Hbbth3/+ mice. The murine Hbbth3/+ phenotype demonstrates mild to moderate anemia and exhibits consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Our results confirmed that chronic apoTf administration resulted in normalization of anemia. Furthermore, we demonstrate normalization of tissue iron content in liver, kidney and heart and attenuation of early tissue changes in non-transfused Hbbth3/+ mice. ApoTf treatment was also found to attenuate transfusion mediated increases in plasma NTBI and asscociated excess tissue iron loading. These therapeutic effects were associated with normalization of Tf saturation and suppressed plasma NTBI. ApoTf treatment was found to modulate a fundamental iron regulatory pathway as evidenced by decreased erythroid Fam132b (erythroferrone) expression, increased liver HAMP expression and plasma hepcidin-25 lelvels and consequently reduced intestinal ferroportin-1 in apoTf treated thalassemic mice