CXCL8 and its cognate receptors CXCR1/CXCR2 in primary myelofibrosis.

BCR::ABL1 negative myeloproliferative neoplasms (MPNs) form a distinct group of hematologic malignancies characterized by sustained proliferation of cells from multiple myeloid lineages. With a median survival of 16-35 months in patients with high-risk disease, primary myelofibrosis (PMF) is considered the most aggressive entity amongst all BCR::ABL1 MPNs. Additionally, a significant subset of patients evolves into secondary acute myeloid leukemia (AML) which has an even poorer prognosis compared to de novo AML. As the exact mechanisms of disease development and progression remain to be elucidated, current therapeutic approaches fail to prevent disease progression or transformation into secondary AML. As each MPN entity is characterized by sustained activation of various immune cells and raised cytokine concentrations within bone marrow and peripheral blood, MPNs may be considered as typical inflammation-related malignancies. However, the exact role and consequences of increased cytokine concentrations within bone marrow and peripheral blood plasma are currently incompletely established. Upregulated cytokines can stimulate cellular proliferation or contribute to the development of an inflammation-related bone marrow niche resulting in genotoxicity and thereby supporting mutagenesis. The neutrophil chemoattractant CXCL8 is of specific interest as its concentration is increased within peripheral blood and bone marrow plasma of patients with PMF. Increased concentration of CXCL8 negatively correlates with overall survival. Furthermore, blockage of the CXCR1/2 axis appears to be able to reduce bone marrow fibrosis and megakaryocyte dysmorphia in murine models. Within this review, we summarize available evidence on the role of the CXCL8-CXCR1/2 axis within the pathogenesis of PMF and discuss potential therapeutic modalities targeting either CXCL8 or its cognate receptors CXCR1/2.

Natural carboxyterminal truncation of human CXCL10 attenuates glycosaminoglycan binding, CXCR3A signaling and lymphocyte chemotaxis, while retaining angiostatic activity.

BACKGROUND: Interferon-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1-73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes. METHODS: Relative levels of CXCL10(1-73) and intact CXCL10(1-77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1-73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1-73) was compared to intact CXCL10(1-77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cell migration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1-73) was also evaluated. RESULTS: Natural CXCL10(1-73) was more abundantly present compared to intact CXCL10(1-77) in synovial fluids of patients with RA. CXCL10(1-73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A. Moreover, CXCL10(1-73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1-73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. CONCLUSION: Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis.

Frequent Allergic Sensitization to Farmed Edible Insects in Exposed Employees.

BACKGROUND: Exposure to insects used in pet food, scientific research, or live fish bait can cause an occupational allergy. The recent shift toward enhanced insect production for human consumption and animal feed will likely expose more employees. OBJECTIVE: To investigate sensitization and symptoms in employees exposed to edible insects in Flanders. METHODS: Fifteen insect-exposed employees were recruited and sensitization was explored by skin prick test, basophil activation test, and immunoblotting. Lung function, FeNO, histamine provocation, and sputum induction were studied. Airborne dust sampling was performed and proteins were studied by silver stain and immunoblotting. RESULTS: Sixty percent of employees self-reported upper respiratory tract symptoms related to insect exposure. Ten employees (71.4%) had a positive histamine provocation test concentration causing a 20% drop in FEV1 less than 8 mg/mL and four (26.7%) had FeNO levels above 25 ppb. Four employees (30.7%) had a positive skin prick test for at least one insect, and seven (58.3%) had a positive basophil activation test. In eight participants with insect sensitization, four (50%) had co-occurring house dust mite sensitization. Two participants had strong IgE binding to a 50-kDa migratory locust allergen, one to a 25-kDa mealworm allergen, and one to mealworm α-amylase. In one center, facility adjustment resulted in a substantial decrease in the inhalable dust fraction. CONCLUSIONS: Insect exposure leads to high levels of sensitization among employees. Most employees reported symptoms of the upper respiratory system, and two-thirds of employees had bronchial hyperreactivity. Prevention and health surveillance will be important in the developing insect-rearing industry.

Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients.

The human chemokine stromal cell-derived factor-1 (SDF-1) or CXCL12 is involved in several homeostatic processes and pathologies through interaction with its cognate G protein-coupled receptor CXCR4. Recent research has shown that CXCL12 is present in the lungs and circulation of patients with coronavirus disease 2019 (COVID-19). However, the question whether the detected CXCL12 is bioactive was not addressed. Indeed, the activity of CXCL12 is regulated by NH2- and COOH-terminal post-translational proteolysis, which significantly impairs its biological activity. The aim of the present study was to characterize proteolytic processing of CXCL12 in broncho-alveolar lavage (BAL) fluid and blood plasma samples from critically ill COVID-19 patients. Therefore, we optimized immunosorbent tandem mass spectrometry proteoform analysis (ISTAMPA) for detection of CXCL12 proteoforms. In patient samples, this approach uncovered that CXCL12 is rapidly processed by site-specific NH2- and COOH-terminal proteolysis and ultimately degraded. This proteolytic inactivation occurred more rapidly in COVID-19 plasma than in COVID-19 BAL fluids, whereas BAL fluid samples from stable lung transplantation patients and the non-affected lung of lung cancer patients (control groups) hardly induced any processing of CXCL12. In COVID-19 BAL fluids with high proteolytic activity, processing occurred exclusively NH2-terminally and was predominantly mediated by neutrophil elastase. In low proteolytic activity BAL fluid and plasma samples, NH2- and COOH-terminal proteolysis by CD26 and carboxypeptidases were observed. Finally, protease inhibitors already approved for clinical use such as sitagliptin and sivelestat prevented CXCL12 processing and may therefore be of pharmacological interest to prolong CXCL12 half-life and biological activity in vivo.

Fast and furious: The neutrophil and its armamentarium in health and disease.

Neutrophils are powerful effector cells leading the first wave of acute host-protective responses. These innate leukocytes are endowed with oxidative and nonoxidative defence mechanisms, and play well-established roles in fighting invading pathogens. With microbicidal weaponry largely devoid of specificity and an all-too-well recognized toxicity potential, collateral damage may occur in neutrophil-rich diseases. However, emerging evidence suggests that neutrophils are more versatile, heterogeneous, and sophisticated cells than initially thought. At the crossroads of innate and adaptive immunity, neutrophils demonstrate their multifaceted functions in infectious and noninfectious pathologies including cancer, autoinflammation, and autoimmune diseases. Here, we discuss the kinetics of neutrophils and their products of activation from bench to bedside during health and disease, and provide an overview of the versatile functions of neutrophils as key modulators of immune responses and physiological processes. We focus specifically on those activities and concepts that have been validated with primary human cells.

Acute-serum amyloid A and A-SAA-derived peptides as formyl peptide receptor (FPR) 2 ligands.

Originally, it was thought that a single serum amyloid A (SAA) protein was involved in amyloid A amyloidosis, but in fact, SAA represents a four-membered family wherein SAA1 and SAA2 are acute phase proteins (A-SAA). SAA is highly conserved throughout evolution within a wide range of animal species suggestive of an important biological function. In fact, A-SAA has been linked to a number of divergent biological activities wherein a number of these functions are mediated via the G protein-coupled receptor (GPCR), formyl peptide receptor (FPR) 2. For instance, through the activation of FPR2, A-SAA has been described to regulate leukocyte activation, atherosclerosis, pathogen recognition, bone formation and cell survival. Moreover, A-SAA is subject to post-translational modification, primarily through proteolytic processing, generating a range of A-SAA-derived peptides. Although very little is known regarding the biological effect of A-SAA-derived peptides, they have been shown to promote neutrophil and monocyte migration through FPR2 activation via synergy with other GPCR ligands namely, the chemokines CXCL8 and CCL3, respectively. Within this review, we provide a detailed analysis of the FPR2-mediated functions of A-SAA. Moreover, we discuss the potential role of A-SAA-derived peptides as allosteric modulators of FPR2.

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.

The chemokines CXCL8 and CXCL12: molecular and functional properties, role in disease and efforts towards pharmacological intervention.

Chemokines are an indispensable component of our immune system through the regulation of directional migration and activation of leukocytes. CXCL8 is the most potent human neutrophil-attracting chemokine and plays crucial roles in the response to infection and tissue injury. CXCL8 activity inherently depends on interaction with the human CXC chemokine receptors CXCR1 and CXCR2, the atypical chemokine receptor ACKR1, and glycosaminoglycans. Furthermore, (hetero)dimerization and tight regulation of transcription and translation, as well as post-translational modifications further fine-tune the spatial and temporal activity of CXCL8 in the context of inflammatory diseases and cancer. The CXCL8 interaction with receptors and glycosaminoglycans is therefore a promising target for therapy, as illustrated by multiple ongoing clinical trials. CXCL8-mediated neutrophil mobilization to blood is directly opposed by CXCL12, which retains leukocytes in bone marrow. CXCL12 is primarily a homeostatic chemokine that induces migration and activation of hematopoietic progenitor cells, endothelial cells, and several leukocytes through interaction with CXCR4, ACKR1, and ACKR3. Thereby, it is an essential player in the regulation of embryogenesis, hematopoiesis, and angiogenesis. However, CXCL12 can also exert inflammatory functions, as illustrated by its pivotal role in a growing list of pathologies and its synergy with CXCL8 and other chemokines to induce leukocyte chemotaxis. Here, we review the plethora of information on the CXCL8 structure, interaction with receptors and glycosaminoglycans, different levels of activity regulation, role in homeostasis and disease, and therapeutic prospects. Finally, we discuss recent research on CXCL12 biochemistry and biology and its role in pathology and pharmacology.

"Glyco-sulfo barcodes" regulate chemokine receptor function.

Chemokine ligands and receptors regulate the directional migration of leukocytes. Post-translational modifications of chemokine receptors including O-glycosylation and tyrosine sulfation have been reported to regulate ligand binding and resulting signaling. Through in silico analyses, we determined potential conserved O-glycosylation and sulfation sites on human and murine CC chemokine receptors. Glyco-engineered CHO cell lines were used to measure the impact of O-glycosylation on CC chemokine receptor CCR5, while mutation of tyrosine residues and treatment with sodium chlorate were performed to determine the effect of tyrosine sulfation. Changing the glycosylation or tyrosine sulfation on CCR5 reduced the receptor signaling by the more positively charged CCL5 and CCL8 more profoundly compared to the less charged CCL3. The loss of negatively charged sialic acids resulted only in a minor effect on CCL3-induced signal transduction. The enzymes GalNAc-T1 and GalNAc-T11 were shown to be involved in the process of chemokine receptor O-glycosylation. These results indicate that O-glycosylation and tyrosine sulfation are involved in the fine-tuning and recognition of chemokine interactions with CCR5 and the resulting signaling.

Sputum from patients with primary ciliary dyskinesia contains high numbers of dysfunctional neutrophils and inhibits efferocytosis.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by recurrent airway infection and inflammation. There is no cure for PCD and to date there are no specific treatments available. Neutrophils are a crucial part of the immune system and are known to be dysfunctional in many inflammatory diseases. So far, the role of the neutrophils in PCD airways is largely unknown. The purpose of this study was to investigate the phenotype and function of airway neutrophils in PCD, and compare them to blood neutrophils. METHODS: Paired peripheral blood and spontaneously expectorated sputum samples from patients with PCD (n = 32) and a control group of patients with non-PCD, non-cystic fibrosis bronchiectasis (n = 5) were collected. The expression of neutrophil-specific surface receptors was determined by flow cytometry. Neutrophil function was assessed by measuring the extent of actin polymerization, production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs) in response to activating stimuli. RESULTS: Sputum neutrophils displayed a highly activated phenotype and were unresponsive to stimuli that would normally induce ROS production, actin polymerization and the expulsion of NETs. In addition, PCD sputum displayed high activity of neutrophil elastase, and impaired the efferocytosis by healthy donor macrophages. CONCLUSIONS: Sputum neutrophils in PCD are dysfunctional and likely contribute to ongoing inflammation in PCD airways. Further research should focus on anti-inflammatory therapies and stimulation of efferocytosis as a strategy to treat PCD.

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.

The GAG-Binding Peptide MIG30 Protects against Liver Ischemia-Reperfusion in Mice.

Ischemia-reperfusion injury (IRI) drives graft rejection and is the main cause of mortality after liver transplantation. During IRI, an intense inflammatory response marked by chemokine production and neutrophil recruitment occurs. However, few strategies are available to restrain this excessive response. Here, we aimed to interfere with chemokine function during IRI in order to disrupt neutrophil recruitment to the injured liver. For this, we utilized a potent glycosaminoglycan (GAG)-binding peptide containing the 30 C-terminal amino acids of CXCL9 (MIG30) that is able to inhibit the binding of chemokines to GAGs in vitro. We observed that mice subjected to IRI and treated with MIG30 presented significantly lower liver injury and dysfunction as compared to vehicle-treated mice. Moreover, the levels of chemokines CXCL1, CXCL2 and CXCL6 and of proinflammatory cytokines TNF-α and IL-6 were significantly reduced in MIG30-treated mice. These events were associated with a marked inhibition of neutrophil recruitment to the liver during IRI. Lastly, we observed that MIG30 is unable to affect leukocytes directly nor to alter the stimulation by either CXCL8 or lipopolysaccharide (LPS), suggesting that its protective properties derive from its ability to inhibit chemokine activity in vivo. We conclude that MIG30 holds promise as a strategy to treat liver IRI and inflammation.

HIV protease inhibitors Nelfinavir and Lopinavir/Ritonavir markedly improve lung pathology in SARS-CoV-2-infected Syrian hamsters despite lack of an antiviral effect.

Nelfinavir is an HIV protease inhibitor that has been widely prescribed as a component of highly active antiretroviral therapy, and has been reported to exert in vitro antiviral activity against SARS-CoV-2. We here assessed the effect of Nelfinavir in a SARS-CoV-2 infection model in hamsters. Despite the fact that Nelfinavir, [50 mg/kg twice daily (BID) for four consecutive days], did not reduce viral RNA load and infectious virus titres in the lung of infected animals, treatment resulted in a substantial improvement of SARS-CoV-2-induced lung pathology. This was accompanied by a dense infiltration of neutrophils in the lung interstitium which was similarly observed in non-infected hamsters. Nelfinavir resulted also in a marked increase in activated neutrophils in the blood, as observed in non-infected animals. Although Nelfinavir treatment did not alter the expression of chemoattractant receptors or adhesion molecules on human neutrophils, in vitro migration of human neutrophils to the major human neutrophil attractant CXCL8 was augmented by this protease inhibitor. Nelfinavir appears to induce an immunomodulatory effect associated with increasing neutrophil number and functionality, which may be linked to the marked improvement in SARS-CoV-2 lung pathology independent of its lack of antiviral activity. Since Nelfinavir is no longer used for the treatment of HIV, we studied the effect of two other HIV protease inhibitors, namely the combination Lopinavir/Ritonavir (Kaletra™) in this model. This combination resulted in a similar protective effect as Nelfinavir against SARS-CoV2 induced lung pathology in hamsters.

Neutrophils in malaria: The good, the bad or the ugly?

Neutrophils are the most abundant circulating leukocytes in human peripheral blood. They are often the first cells to respond to an invading pathogen and might therefore play an important role in malaria. Malaria is a globally important disease caused by Plasmodium parasites, responsible for more than 400,000 deaths each year. Most of these deaths are caused by complications, including cerebral malaria, severe malarial anaemia, placental malaria, renal injury, metabolic problems and malaria-associated acute respiratory distress syndrome. Neutrophils contribute in the immune defence against malaria, through clearance of parasites via phagocytosis, production of reactive oxygen species and release of neutrophil extracellular traps (NETs). However, Plasmodium parasites diminish antibacterial functions of neutrophils, making patients more susceptible to other infections. Neutrophils might also be involved in the development of malaria complications, for example via the release of toxic granules and NETs. However, technical pitfalls in the determination of the roles of neutrophils have caused contradicting results. Further investigations need to consider these pitfalls, in order to elucidate the role of neutrophils in malaria complications.

Method Matters: Effect of Purification Technology on Neutrophil Phenotype and Function.

Neutrophils are the most abundant leukocytes in human blood and the first cells responding to infection and injury. Due to their limited ex vivo lifespan and the impossibility to cryopreserve or expand them in vitro, neutrophils need to be purified from fresh blood for immediate use in experiments. Importantly, neutrophil purification methods may artificially modify the phenotype and functional characteristics of the isolated cells. The aim of this study was to expose the effects of 'classical' density-gradient purification versus the more expensive but faster immunomagnetic isolation on neutrophil phenotype and functionality. We found that in the absence of inflammatory stimuli, density-gradient-derived neutrophils showed increased polarization responses as well as enhanced release of reactive oxygen species (ROS), neutrophil extracellular traps (NETs) and granular proteins compared to cells derived from immunomagnetic isolation, which yields mostly quiescent neutrophils. Upon exposure to pro-inflammatory mediators, immunomagnetic isolation-derived neutrophils were significantly more responsive in polarization, ROS production, phagocytosis, NETosis and degranulation assays, in comparison to density-gradient-derived cells. We found no difference in chemotactic response in Multiscreen and under-agarose migration assays, but Boyden assays showed reduced chemotaxis of immunomagnetic isolation-derived neutrophils. Finally, we confirmed that density-gradient purification induces artificial activation of neutrophils, evidenced by e.g. higher expression of CD66b, formyl peptide receptor 1 (FPR1) and CD35, and the appearance of a separate neutrophil population expressing surface molecules atypical for neutrophils (e.g. CXCR3, MHC-II and CD14). Based on these results, we recommend using immunomagnetic separation of neutrophils for studying neutrophil polarization, phagocytosis, ROS production, degranulation and NETosis, whereas for Boyden chemotaxis assays, the density-gradient purification is more suitable.

Reported Cases and Diagnostics of Occupational Insect Allergy: A Systematic Review.

A significant part of adult-onset asthma is caused by occupational exposure to both high- and low-molecular-mass agents. Insects are occasionally described to cause occupational allergy in professions including anglers and fishers, laboratory workers, employees of aquaculture companies, farmers, bakers, sericulture workers and pet shop workers. Occupational insect allergies are often respiratory, causing asthma or rhinoconjunctivitis, but can be cutaneous as well. The European Union recently approved three insect species for human consumption, enabling an industry to develop where more employees could be exposed to insect products. This review overviews knowledge on occupational insect allergy risks and the tools used to diagnose employees. Despite the limited availability of commercial occupational insect allergy diagnostics, 60.9% of 164 included reports used skin prick tests and 63.4% of reports used specific IgE tests. In 21.9% of reports, a more elaborate diagnosis of occupational asthma was made by specific inhalation challenges or peak expiratory flow measurements at the workplace. In some work environments, 57% of employees were sensitized, and no less than 60% of employees reported work-related symptoms. Further development and optimization of specific diagnostics, together with strong primary prevention, may be vital to the health conditions of workers in the developing insect industry.

Atypical response to bacterial coinfection and persistent neutrophilic bronchoalveolar inflammation distinguish critical COVID-19 from influenza.

Neutrophils are recognized as important circulating effector cells in the pathophysiology of severe coronavirus disease 2019 (COVID-19). However, their role within the inflamed lungs is incompletely understood. Here, we collected bronchoalveolar lavage (BAL) fluids and parallel blood samples of critically ill COVID-19 patients requiring invasive mechanical ventilation and compared BAL fluid parameters with those of mechanically ventilated patients with influenza, as a non-COVID-19 viral pneumonia cohort. Compared with those of patients with influenza, BAL fluids of patients with COVID-19 contained increased numbers of hyperactivated degranulating neutrophils and elevated concentrations of the cytokines IL-1ß, IL-1RA, IL-17A, TNF-α, and G-CSF; the chemokines CCL7, CXCL1, CXCL8, CXCL11, and CXCL12α; and the protease inhibitors elafin, secretory leukocyte protease inhibitor, and tissue inhibitor of metalloproteinases 1. In contrast, α-1 antitrypsin levels and net proteolytic activity were comparable in COVID-19 and influenza BAL fluids. During antibiotic treatment for bacterial coinfections, increased BAL fluid levels of several activating and chemotactic factors for monocytes, lymphocytes, and NK cells were detected in patients with COVID-19 whereas concentrations tended to decrease in patients with influenza, highlighting the persistent immunological response to coinfections in COVID-19. Finally, the high proteolytic activity in COVID-19 lungs suggests considering protease inhibitors as a treatment option.

The Antimicrobial Activity of Peripheral Blood Neutrophils Is Altered in Patients with Primary Ciliary Dyskinesia.

The airways of patients with primary ciliary dyskinesia (PCD) contain persistently elevated neutrophil numbers and CXCL8 levels. Despite their abundance, neutrophils fail to clear the airways from bacterial infections. We investigated whether neutrophil functions are altered in patients with PCD. Neutrophils from patients and healthy controls (HC) were isolated from peripheral blood and exposed to various bacterial stimuli or cytokines. Neutrophils from patients with PCD were less responsive to low levels of fMLF in three different chemotaxis assays (p < 0.05), but expression of the fMLF receptors was unaltered. PCD neutrophils showed normal phagocytic function and expression of adhesion molecules. However, PCD neutrophils produced less reactive oxygen species upon stimulation with bacterial products or cytokines compared to HC neutrophils (p < 0.05). Finally, the capacity to release DNA, as observed during neutrophil extracellular trap formation, seemed to be reduced in patients with PCD compared to HC (p = 0.066). These results suggest that peripheral blood neutrophils from patients with PCD, in contrast to those of patients with cystic fibrosis or COPD, do not show features of over-activation, neither on baseline nor after stimulation. If these findings extend to lung-resident neutrophils, the reduced neutrophil activity could possibly contribute to the recurrent respiratory infections in patients with PCD.

Kinetics of peripheral blood neutrophils in severe coronavirus disease 2019.

OBJECTIVES: Emerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID-19). We isolated neutrophils from the blood of COVID-19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation. METHODS: Multicolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co-culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) allowed us to investigate viral replication in neutrophils. RESULTS: Upon ICU admission, patients displayed high plasma concentrations of granulocyte-colony-stimulating factor (G-CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10-, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non-metalloproteinase-derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID-19 neutrophils were hyper-responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS-CoV-2 failed to replicate inside human neutrophils. CONCLUSION: Our study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID-19 patients, and supports the concept of an increased neutrophil activation state in the circulation.