Automated, Point-of-Care mobile flow cytometry: Bringing the laboratory to the sample.

Background: Innate effector cells are very responsive to infectious and inflammatory cues found in damaged and inflamed tissues. Their activation is a potential target to assess the state of the immune system. Unfortunately, these cells are very susceptible for ex-vivo activation, hampering accurate interpretation of flow cytometry data. Whether a brief window exists before ex-vivo activation starts to occur is currently unknown. Aims: 1) This study extensively investigated ex-vivo activation of innate effector cells over time. 2) We tested the feasibility of applying a mobile, automated, flow cytometry laboratory for out-of-hospital Point-of-Care analyses to minimize ex-vivo activation bias. Methods: 1) Ex-vivo neutrophil, eosinophil and monocyte activation in a blood collection tube over time and the reactivity to a formyl-peptide was investigated in a healthy cohort. 2) To facilitate fast, out-of-hospital analysis, application of the mobile flow cytometry was tested by placing an automated flow cytometer into a van. The stability of the setup was assessed by repetitively measuring laser alignment and fluorescence verification beads. Findings: 1) Immediately after venipuncture activation marker expression on neutrophils, eosinophils and monocyte subsets started to change in a time-dependent manner. 2) The mobile flow cytometry laboratory travelled over 3000 km, performing measurements at 19 locations with a median single-person-set-up time of 14 min. The laser alignment and fluorescence were stable during all experiments. Conclusions: Accurate flow data of innate immune cells are only obtained when ex-vivo activation is kept to minimum. The use of a mobile, fast, automated, flow cytometry laboratory for out-of-hospital Point-of-Care analyses provides new investigational and diagnostic possibilities outside major hospital flow cytometry laboratories.

Point-of-care neutrophil CD64 as a rule in diagnostic test for bacterial infections in the emergency department.

INTRODUCTION: Bacterial infections are frequently seen in the emergency department (ED), but can be difficult to distinguish from viral infections and some non-infectious diseases. Common biomarkers such as c-reactive protein (CRP) and white blood cell (WBC) counts fail to aid in the differential diagnosis. Neutrophil CD64 (nCD64), an IgG receptor, is suggested to be more specific for bacterial infections. This study investigated if nCD64 can distinguish bacterial infections from other infectious and non-infectious diseases in the ED. METHODS: All COVID-19 suspected patients who visited the ED and for which a definitive diagnosis was made, were included. Blood was analyzed using an automated flow cytometer within 2 h after presentation. Patients were divided into a bacterial, viral, and non-infectious disease group. We determined the diagnostic value of nCD64 and compared this to those of CRP and WBC counts. RESULTS: Of the 291 patients presented at the ED, 182 patients were included with a definitive diagnosis (bacterial infection n = 78; viral infection n = 64; non-infectious disease n = 40). ROC-curves were plotted, with AUCs of 0.71 [95%CI: 0.64-0.79], 0.77 [0.69-0.84] and 0.64 [0.55-0.73] for nCD64, WBC counts and CRP, respectively. In the bacterial group, nCD64 MFI was significantly higher compared to the other groups (p < 0.01). A cut-off of 9.4 AU MFI for nCD64 corresponded with a positive predictive value of 1.00 (sensitivity of 0.27, a specificity of 1.00, and an NPV of 0.64). Furthermore, a diagnostic algorithm was constructed which can serve as an example of what a future biomarker prediction model could look like. CONCLUSION: For patients in the ED presenting with a suspected infection, nCD64 measured with automatic flow cytometry, has a high specificity and positive predictive value for diagnosing a bacterial infection. However, a low nCD64 cannot rule out a bacterial infection. For future purposes, nCD64 should be combined with additional tests to form an algorithm that adequately diagnoses infectious diseases.

Quality over quantity; eosinophil activation status will deepen the insight into eosinophilic diseases.

Eosinophil associated diseases have gained much attention recently because of the introduction of specific eosinophil targeted therapies. These diseases range from acute parasitic infections to chronic inflammatory diseases such as eosinophilic asthma. In eosinophilic asthma an increased eosinophil cell count in peripheral blood is the gold standard for determination of the pheno-/endotype and severity of disease. Despite a broad consensus there is concern on validity of this simple measurement, because the eosinophil compartment is far from homogenous. Multiple tissues harbour non-activated cells under homeostatic conditions and other tissues, normally devoid of eosinophils, become infested with these cells under inflammatory conditions. It will, therefore, be clear that eosinophils become differentially (pre)-activated at different tissue sites in homeostatic and inflammatory conditions. This complexity should be investigated in detail as it is 1) far from clear what the long-term side effects are that are caused by application of eosinophil targeted therapies in a "one size fits all" concept and 2) real-world data of eosinophil targeted therapies in asthma shows a broad variety in the treatment response. This review will focus on complex mechanisms of eosinophil activation in vivo to create a better view on the dynamics of the eosinophil compartment in health and disease both to prevent collateral damage caused by aberrant activation of eosinophils ánd to improve effectiveness of eosinophil targeted treatments.

Longitudinal assessment of the inflammatory response: The next step in personalized medicine after severe trauma.

Infections in trauma patients are an increasing and substantial cause of morbidity, contributing to a mortality rate of 5-8% after trauma. With increased early survival rates, up to 30-50% of multitrauma patients develop an infectious complication. Trauma leads to a complex inflammatory cascade, in which neutrophils play a key role. Understanding the functions and characteristics of these cells is important for the understanding of their involvement in the development of infectious complications. Recently, analysis of neutrophil phenotype and function as complex biomarkers, has become accessible for point-of-care decision making after trauma. There is an intriguing relation between the neutrophil functional phenotype on admission, and the clinical course (e.g., infectious complications) of trauma patients. Potential neutrophil based cellular diagnostics include subsets based on neutrophil receptor expression, responsiveness of neutrophils to formyl-peptides and FcγRI (CD64) expression representing the infectious state of a patient. It is now possible to recognize patients at risk for infectious complications when presented at the trauma bay. These patients display increased numbers of neutrophil subsets, decreased responsiveness to fMLF and/or increased CD64 expression. The next step is to measure these biomarkers over time in trauma patients at risk for infectious complications, to guide decision making regarding timing and extent of surgery and administration of (preventive) antibiotics.

Neutrophil and Eosinophil Responses Remain Abnormal for Several Months in Primary Care Patients With COVID-19 Disease.

Introduction: Neutrophil and eosinophil activation and its relation to disease severity has been understudied in primary care patients with COVID-19. In this study, we investigated whether the neutrophil and eosinophil compartment were affected in primary care patients with COVID-19. Methods: COVID-19 patients, aged ≥ 40 years with cardiovascular comorbidity presenting to the general practitioner with substantial symptoms, partaking in the COVIDSat@Home study between January and April 2021, were included. Blood was drawn during and 3 to 6 months after active COVID-19 disease and analyzed by automated flow cytometry, before and after stimulation with a formyl-peptide (fNLF). Mature neutrophil and eosinophil markers at both time points were compared to healthy controls. A questionnaire was conducted on disease symptoms during and 3 to 6 months after COVID-19 disease. Results: The blood of 18 COVID-19 patients and 34 healthy controls was analyzed. During active COVID-19 disease, neutrophils showed reduced CD10 (p = 0.0360), increased CD11b (p = 0.0002) and decreased CD62L expression (p < 0.0001) compared to healthy controls. During active COVID-19 disease, fNLF stimulated neutrophils showed decreased CD10 levels (p < 0.0001). Three to six months after COVID-19 disease, unstimulated neutrophils showed lowered CD62L expression (p = 0.0003) and stimulated neutrophils had decreased CD10 expression (p = 0.0483) compared to healthy controls. Both (un)stimulated CD10 levels increased 3 to 6 months after active disease (p = 0.0120 and p < 0.0001, respectively) compared to during active disease. Eosinophil blood counts were reduced during active COVID-19 disease and increased 3 to 6 months after infection (p < 0.0001). During active COVID-19, eosinophils showed increased unstimulated CD11b (p = 0.0139) and decreased (un)stimulated CD62L expression (p = 0.0036 and p = 0.0156, respectively) compared to healthy controls. Three to six months after COVID-19 disease, (un)stimulated eosinophil CD62L expression was decreased (p = 0.0148 and p = 0.0063, respectively) and the percentage of CD11bbright cells was increased (p = 0.0083 and p = 0.0307, respectively) compared to healthy controls. Conclusion: Automated flow cytometry analysis reveals specific mature neutrophil and eosinophil activation patterns in primary care patients with COVID-19 disease, during and 3 to 6 months after active disease. This suggests that the neutrophil and eosinophil compartment are long-term affected by COVID-19 in primary care patients. This indicates that these compartments may be involved in the pathogenesis of long COVID.

Immunological and hematological effects of IL-5(Rα)-targeted therapy: An overview.

IL-5 is an important cytokine for priming and survival of mature eosinophils and for proliferation and maturation of their progenitors. Hence, IL-5(Rα) targeting will be increasingly used in diseases where eosinophils are the key immune effector cells such as eosinophilic asthma (EA), hypereosinophilic syndrome (HES), eosinophilic esophagitis (EE), and eosinophilic granulomatosis with polyangiitis (EGPA). Therefore, several neutralizing monoclonal antibodies directed against IL-5 (mepolizumab and reslizumab) and its receptor IL-5Rα (benralizumab) have found or will find their way to the clinic. While the clinical effect of these drugs has been extensively investigated and reviewed, the understanding of the underlying immunological and hematological mechanisms remains less clear. This review will discuss the translational outcomes of treatment with these monoclonal antibodies in humans to shed light on the mechanisms underlying the main immunological and hematological findings from these clinical trials in humans.

IL-3 up-regulates and activates human eosinophil CD32 and αMß2 integrin causing degranulation.

BACKGROUND: Eosinophils contribute to the pathogenesis of multiple diseases, including asthma. Treatment with antibodies targeting IL-5 or IL-5 receptor α reduces the frequency of asthma exacerbations. Eosinophil receptors for IL-5 share a common ß-chain with IL-3 and GM-CSF receptors. We recently reported that IL-3 is more potent than IL-5 or GM-CSF in maintaining the ERK/p90S6K/RPS6 ribosome-directed signaling pathway, leading to increased protein translation. OBJECTIVE: We aimed to determine disease-relevant consequences of prolonged eosinophil stimulation with IL-3. RESULTS: Human blood eosinophils were used to establish the impact of activation with IL-3 on IgG-driven eosinophil degranulation. When compared to IL-5, continuing exposure to IL-3 further induced degranulation of eosinophils on aggregated IgG via increased production and activation of both CD32 (low affinity IgG receptor) and αMß2 integrin. In addition, unlike IL-5 or GM-CSF, IL-3 induced expression of CD32B/C (FCGRIIB/C) subtype proteins, without changing CD32A (FCGRIIA) protein and CD32B/C mRNA expression levels. Importantly, these in vitro IL-3-induced modifications were recapitulated in vivo on airway eosinophils. CONCLUSIONS AND CLINICAL RELEVANCE: We observed for the first time upregulation of CD32B/C on eosinophils, and identified IL-3 as a potent inducer of CD32- and αMß2-mediated eosinophil degranulation.

Diagnosing eosinophilic asthma using a multivariate prediction model based on blood granulocyte responsiveness.

BACKGROUND: The identification of inflammatory asthma phenotypes, using sputum analysis, has proven its value in diagnosis and disease monitoring. However due to technical limitations of sputum analysis, there is a strong need for fast and noninvasive diagnostics. This study included the activation state of eosinophils and neutrophils in peripheral blood to phenotype and monitor asthma. OBJECTIVES: To (i) construct a multivariable model using the activation state of blood granulocytes, (ii) compare its diagnostic value with sputum eosinophilia as gold standard and (iii) validate the model in an independent patient cohort. METHODS: Clinical parameters, activation of blood granulocytes and sputum characteristics were assessed in 115 adult patients with asthma (training cohort/Utrecht) and 34 patients (validation cohort/Oxford). RESULTS: The combination of blood eosinophil count, fractional exhaled nitric oxide, Asthma Control Questionnaire, medication use, nasal polyposis, aspirin sensitivity and neutrophil/eosinophil responsiveness upon stimulation with formyl-methionyl-leucyl phenylalanine was found to identify sputum eosinophilia with 90.5% sensitivity and 91.5% specificity in the training cohort and with 77% sensitivity and 71% specificity in the validation cohort (relatively high percentage on oral corticosteroids [OCS]). CONCLUSIONS: The proposed prediction model identifies eosinophilic asthma without the need for sputum induction. The model forms a noninvasive and externally validated test to assess eosinophilic asthma in patients not on OCS.

Genetic variation in uncontrolled childhood asthma despite ICS treatment.

Genetic variation may partly explain asthma treatment response heterogeneity. We aimed to identify common and rare genetic variants associated with asthma that was not well controlled despite inhaled corticosteroid (ICS) treatment. Data of 110 children was collected in the Children Asthma Therapy Optimal trial. Associations of genetic variation with measures of lung function (FEV1%pred), airway hyperresponsiveness (AHR) to methacholine (Mch PD20) and treatment response outcomes were analyzed using the exome chip. The 17q12-21 locus (containing ORMDL3 and GSMDB) previously associated with childhood asthma was investigated separately. Single-nucleotide polymorphisms (SNPs) in the 17q12-21 locus were found nominally associated with the outcomes. The strongest association in this region was found for rs72821893 in KRT25 with FEV1%pred (P=3.75*10(-5)), Mch PD20 (P=0.00095) and Mch PD20-based treatment outcome (P=0.006). No novel single SNPs or burden tests were significantly associated with the outcomes. The 17q12-21 region was associated with FEV1%pred and AHR, and additionally with ICS treatment response.

Elevated mean neutrophil volume represents altered neutrophil composition and reflects damage after myocardial infarction.

Myocardial infarction (MI) induces an inflammatory response in which neutrophils fulfill a prominent role. Mean neutrophil volume (MNV) represents the average size of the circulating neutrophil population. Our goal was to determine the effect of MI on MNV and investigate the mechanisms behind MNV elevation. MNV of 84 MI patients was compared with the MNV of 209 stable angina patients and correlated to simultaneously measured CK levels. Fourteen pigs were subjected to temporary coronary balloon occlusion and blood was sampled at multiple time points to measure MNV. Echocardiography was performed followed by ex vivo infarct size assessment after 72 h. MNV was higher in MI patients compared to stable angina patients (602 SD26 AU vs. 580 SD20 AU, p < 0.0001) and correlated with simultaneously measured CK levels (R = 0.357, p < 0.0001). In pigs, MNV was elevated post-MI (451 SD11 AU vs. 469 SD12 AU), p < 0.0001). MNV correlated with infarct size (R = 0.705, p = 0.007) and inversely correlated with left ventricular ejection fraction (R = -0.718, p = 0.009). Cell sorting revealed an increased presence of banded neutrophils after MI, which have a higher MNV compared to mature neutrophils post-MI (495 SD14 AU vs. 478 SD11 AU, p = 0.012). MNV from coronary sinus blood was higher than MNV of neutrophils from simultaneously sampled arterial blood (463 SD7.6 AU vs. 461 SD8.6 AU, p = 0.013) post-MI. The current study shows MNV is elevated and reflects cardiac damage post-MI. MNV increases due to altered neutrophil composition and systemic neutrophil activation. MNV may be an interesting parameter for prognostic assessment in MI and provide new insights into pathological innate immune responses evoked by ischemia-reperfusion.

Similar activation state of neutrophils in sputum of asthma patients irrespective of sputum eosinophilia.

Inflammatory phenotypes of asthma are associated with differences in disease characteristics. It is unknown whether these inflammatory phenotypes are reflected by the activation status of neutrophils in blood and sputum. We obtained peripheral blood and induced sputum from 21 asthma patients and stratified our samples based on sputum eosinophilia resulting in two groups (>3% eosinophils: n = 13, <3%: n = 8). Eosinophils and neutrophils from blood and sputum were analysed for expression of activation and degranulation markers by flow cytometry. Data were analysed by both classical, non-parametric statistics and a multi-dimensional approach, using principal component analysis (PCA). Patients with sputum eosinophilia were characterized by increased asthma control questionnaire (ACQ) scores and blood eosinophil counts. Both sputum neutrophils and eosinophils displayed an activated and degranulated phenotype compared to cells obtained from blood. Specifically, degranulation of all granule types was detected in sputum cells, combined with an increased expression of the activation markers (activated) Mac-1 (CD11b), programmed death ligand 1 (PD-L1) (CD274) and a decreased expression of CD62L. CD69 expression was only increased on sputum eosinophils. Surface marker expression of neutrophils was similar in the presence or absence of eosinophilia, either by single or multi-dimensional analysis. Sputum neutrophils were highly activated and degranulated irrespective of sputum eosinophilia. Therefore, we conclude that differences in granulocyte activation in sputum and/or blood are not associated with clinical differences in the two groups of asthma patients. The finding of PD-L1 expression on sputum granulocytes suggests an immunomodulatory role of these cells in the tissue.

ST13 polymorphisms and their effect on exacerbations in steroid-treated asthmatic children and young adults.

BACKGROUND: The clinical response to inhaled corticosteroids (ICS) is associated with single nucleotide polymorphisms (SNPs) in various genes. This study aimed to relate variations in genes in the steroid pathway and asthma susceptibility genes to exacerbations in children and young adults treated with ICS. METHODS: We performed a meta-analysis of three cohort studies: Pharmacogenetics of Asthma Medication in Children: Medication with Anti-Inflammatory effects (n = 357, age: 4-12 years, the Netherlands), BREATHE (n = 820, age: 3-22 years, UK) and Paediatric Asthma Gene Environment Study (n = 391, age: 2-16 years, UK). Seventeen genes were selected based on a role in the glucocorticoid signalling pathway or a reported association with asthma. Two outcome parameters were used to reflect exacerbations: hospital visits and oral corticosteroid (OCS) use in the previous year. The most significant associations were tested in three independent validation cohorts; the Childhood Asthma Management Programme (clinical trial, n = 172, age: 5-12 years, USA), the Genes- environment and Mixture in Latino Americans II- study (n = 745, age: 8-21, USA) and the Pharmacogenetics of adrenal suppression cohort (n = 391, age: 5-18, UK) to test the robustness of the findings. Finally, all results were meta-analysed. RESULTS: Two SNPs in ST13 (rs138335 and rs138337), but not in the other genes, were associated at a nominal level with an increased risk of exacerbations in asthmatics using ICS in the three cohorts studied. In a meta-analysis of all six studies, ST13 rs138335 remained associated with an increased risk of asthma-related hospital visits and OCS use in the previous year; OR = 1.22 (P = 0.013) and OR = 1.22 (P = 0.0017), respectively. CONCLUSION AND CLINICAL RELEVANCE: A novel susceptibility gene, ST13, coding for a cochaperone of the glucocorticoid receptor, is associated with exacerbations in asthmatic children and young adults despite their ICS use. Genetic variation in the glucocorticoid signalling pathway may contribute to the interindividual variability in clinical response to ICS treatment in children and young adults.

Acute-phase concentrations of soluble fibrinogen inhibit neutrophil adhesion under flow conditions in vitro through interactions with ICAM-1 and MAC-1 (CD11b/CD18).

BACKGROUND: Immobilized fibrinogen and fibrin facilitate leukocyte adhesion, as they are potent ligands for leukocyte MAC-1 (CD11b/CD18). However, fibrinogen in its soluble form also binds to MAC-1, albeit with low affinity. The level of soluble fibrinogen is increased during chronic and acute inflammation, but the function of this increase is unknown. OBJECTIVES: To study the effect of soluble fibrinogen in concentrations found in severe acute inflammation on leukocyte adhesion. METHODS: Isolated leukocytes and soluble fibrinogen were studied in various in vitro settings under static and under flow conditions. RESULTS: Soluble fibrinogen functioned as a natural antagonist of neutrophil functions that are dependent on MAC-1, such as the respiratory burst induced by unopsonized zymosan and adhesion to ICAM-1 and heparin. In addition, soluble fibrinogen inhibited lymphocyte function-associated antigen 1-dependent lymphocyte binding to ICAM-1 through a direct interaction with ICAM-1. Soluble fibrinogen reduced MAC-1-dependent binding of interleukin-8-activated neutrophils to ICAM-1-expressing cells under flow conditions. Importantly soluble fibrinogen in acute-phase concentrations (4-10 mg mL(-1) ) dose-dependently reduced neutrophil firm adhesion to tumor necrosis factor-α-activated endothelium to 40% under flow conditions. CONCLUSIONS: We propose a model in which the increased circulating concentrations of soluble fibrinogen found during the acute-phase response can act as a natural antagonist of leukocyte recruitment, and therefore might contribute to the resolution of inflammation.

Lipopolysaccharide and hemorrhagic shock cause systemic inflammation by different mechanisms.

BACKGROUND: Hemorrhagic shock (HS) and sepsis are common after trauma. These trauma patients often need ventilatory support. The resulting hyperinflammatory state can cause neutrophil-mediated complications such as adult respiratory distress syndrome. An important underlying mechanism is polymorphonuclear neutrophil (PMN) priming by damage-associated molecular patterns (DAMPs, caused by, e.g., HS and ventilation) and by pathogen-associated molecular patterns (PAMPs, e.g., lipopolysaccharide [LPS] in sepsis). The aim of this study was to compare the inflammatory response induced by DAMPs (liberated during HS) and PAMPs (LPS challenge) under conditions of high-volume ventilation. METHODS: Twenty-seven male Sprague-Dawley rats were randomized for mechanical ventilation (MV) alone (9 rats; positive end-expiratory pressure, 5 cm H2O; pressure control, +20 cm H2O; FIO2, 0.33), MV + HS (9 rats; hemorrhage, 30% volume loss) or MV + LPS (9 rats, LPS, 5mg/kg intravenously administered). Five rats were used as controls. Total PMN count and differentials were determined. In addition, the expression of activation markers (CD62L and CD11b) on blood-derived PMNs was measured by flow cytometry. Pulmonary inflammatory response was measured by PMN counts in bronchoalveolar fluid. The presence of neutrophils in the lung was determined by total myeloperoxidase. Results are expressed as means ± SEM; p ≤ 0.05 is considered statistically significant. RESULTS: All treated rats had more neutrophils in the blood and bronchoalveolar fluid compared with the controls. Myeloperoxidase was significantly higher in all groups compared with controls. MV and MV + HS rats had more blood neutrophils with CD62L(bright)/CD11b(dim) phenotypes, whereas MV + LPS rats showed more CD62L(dim)/CD11b(bright) neutrophils, indicative for activated cells. CONCLUSION: All ventilated rats showed a systemic inflammatory response. The responses to HS and MV were very similar, suggesting a common pathway of DAMP-associated inflammation. In marked contrast, LPS showed a different neutrophil phenotype (CD62L(dim)/CD11b(bright)), suggesting a different inflammatory response. It is concluded that shock induced by DAMPs and PAMPs have different underlying mechanisms.

Isolated blunt chest injury leads to transient activation of circulating neutrophils.

INTRODUCTION: The acute respiratory distress syndrome (ARDS) is a severe and frequently seen complication in multi-trauma patients. ARDS is caused by an excessive innate immune response with a clear role for neutrophils. As ARDS is more frequently seen in trauma patients with chest injury, we investigated the influence of chest injury on the systemic neutrophil response and the development of ARDS. MATERIALS AND METHODS: Thirteen patients with isolated blunt chest injury [abbreviated injury score (AIS) 2-5] were included. To avoid systemic inflammation caused by tissue damage outside the thorax, injuries to other regions than the chest did not exceed an AIS of 2. At 3, 9 and 24 h after injury, the expression of circulating activating molecules on neutrophils and levels of circulating interleukine (IL)-6 were determined. Blood samples from eight healthy volunteers were used as control. RESULTS: Blunt chest injury resulted in the activation of circulating neutrophils, as characterized by a decreased expression of l-selectin (CD62L), CXCR2 (CD182b) and C5aR (CD88) compared to control (p < 0.05). Expression of l-selectin, CXCR2 and C5aR was partially restored at 24 h after injury. In addition, the mean expression of FcγRIII (CD16) dropped (p < 0.001), indicating the recruitment of young neutrophils into the circulation. IL-6 levels increased to a maximum mean concentration of 86 ± 31 pg/ml at 24 h postinjury. None of the patients developed ARDS. CONCLUSION: Blunt chest trauma caused a systemic inflammatory reaction with transient activation of neutrophils and mobilization of young neutrophils into the circulation. Isolated chest injury, however, was not abundant enough to cause ARDS, so a second hit appears crucial.

Biomarkers of therapy responsiveness in asthma: pitfalls and promises.

Asthma is one of the most common chronic diseases worldwide. There is a large inter-individual variability in response to asthma treatment. Most patients respond well to standard therapy; however, a small proportion of the patients remain symptomatic despite treatment with high dosages of corticosteroids. Uncontrolled asthma leads to a decreased quality of life. Therefore, it is important to identify individuals who will respond poorly to standard asthma medication, especially to standard maintenance therapy with inhaled corticosteroids, at an early stage. Response to anti-inflammatory therapy is generally monitored by the assessment of clinical symptoms, which only partially correlates with underlying airway inflammation. The identification of specific inflammatory biomarkers might help to guide treatment or predict a corticosteroid response more accurately. Some inflammatory biomarkers are already finding their way into clinical practice (e.g. fraction of nitric oxide in exhaled breath), whereas others are predominantly used as a research tool (e.g. profiles of volatile organic compounds). Currently, there is no inflammatory biomarker used in routine clinical practice to predict a corticosteroid response. More knowledge on the underlying biological mechanism(s) of heterogeneous therapeutic responses could help to identify novel biomarkers. This review will focus on inflammatory patterns and genetic variations that may underlie differences in treatment response in patients with asthma, and will provide an overview of inflammatory biomarkers that could potentially serve as response predictors.

Steroids induce a disequilibrium of secreted interleukin-1 receptor antagonist and interleukin-1ß synthesis by human neutrophils.

Chronic obstructive pulmonary disease (COPD) is characterised by neutrophilic inflammation in the airways and these neutrophils contribute to the production of inflammatory mediators. Dampening the production of proinflammatory mediators might be an important strategy to treat COPD and glucocorticosteroids are known to do so via inhibition of nuclear factor-κB. However, this pathway is important for the control of pro- and anti-inflammatory genes. We studied the effects of dexamethasone on production and secretion of pro-inflammatory interleukin (IL)-1ß and anti-inflammatory secreted IL-1 receptor antagonist (sIL-1Ra) by human neutrophils activated with tumor necrosis factor (TNF)-α. In vitro, TNF-α-stimulated neutrophils produced significant amounts of IL-1ß and sIL-1Ra; this production was inhibited by dexamethasone. However, synthesis and secretion of sIL-1Ra was inhibited at lower concentrations dexamethasone compared to IL-1ß, which changed the IL-1ß:sIL-1Ra ratio significantly. This altered ratio resulted in a more pro-inflammatory condition, as visualised by increased intercellular adhesion molecule-1 expression on human endothelial cells. In vivo, moderate-to-severe COPD patients using inhaled glucocorticosteroids have decreased plasma sIL-Ra levels compared with mild-to-moderate patients not on glucocorticosteroid treatment. In conclusion, dexamethasone induces a pro-inflammatory shift in the IL-1ß:sIL-1Ra cytokine balance in neutrophils in vitro, which might contribute to a lack of endogenous anti-inflammatory signals to dampen inflammation in vivo.

Human neutrophils switch to an activated phenotype after homing to the lung irrespective of inflammatory disease.

Systemic inflammation can be investigated by changes in expression profiles of neutrophil receptors. Application of this technology for analysis of neutrophil phenotypes in diseased tissues is hampered by the absence of information regarding the modulation of neutrophil phenotypes after extravasation to tissues under non-inflammatory conditions. To fill this gap we measured the expression of neutrophil receptors in bronchoalveolar lavage fluid (BALF) and in the peripheral blood of healthy volunteers, which included both smokers and non-smokers. Blood and BALF neutrophils were identified by CD16(bright)/CD45(dim) cells, and triple-stained with antibodies directed against integrins, chemokine- and Fc gamma-receptors. BALF neutrophils of healthy volunteers showed an activated phenotype characterized by Mac-1 (CD11b)(bright), L-selectin (CD62L)(dim), intercellular adhesion molecule 1 (ICAM-1) (CD54)(bright), Fc gamma RII (CD32)(bright), C5a receptor (CD88)(bright) and CD66b(bright). A similar phenotype was observed for BALF neutrophils of patients affected by sarcoidosis. Furthermore, our results demonstrate a modulated expression of C5a receptor (CD88) and ICAM-1 (CD54) in neutrophils of sarcoidosis patients. In conclusion, our data indicate that neutrophils found in the lung exhibit an activated phenotype under both homeostatic and inflammatory conditions.