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Abstract. Objectives:. The number of geriatric hip fracture patients is high and expected to rise in the coming years, and many are frail and at risk for adverse outcomes. Early identification of high-risk patients is crucial to balance treatment and optimize outcome, but remains challenging. Previous research in patients with multitrauma suggested that neutrophil phenotype analysis could aid in early identification of high-risk patients. This pilot study investigated the feasibility and clinical value of neutrophil phenotype analysis in geriatric patients with a hip fracture. Methods:. A prospective study was conducted in a regional teaching hospital in the Netherlands. At the emergency department, blood samples were collected from geriatric patients with a hip fracture and analyzed using automated flow cytometry. Flow cytometry data were processed using an automated clustering algorithm. Neutrophil activation data were compared with a healthy control cohort. Neutrophil phenotype categories were assessed based on two-dimensional visual assessment of CD16/CD62L expression. Results:. Blood samples from 45 geriatric patients with a hip fracture were included. Neutrophils showed an increased activation profile and decreased responsiveness to formyl peptides when compared to healthy controls. The neutrophil phenotype of all patients was categorized. The incidence of severe adverse outcome was significantly different between the different categories (P = 0.0331). Moreover, patients with neutrophil phenotype category 0 developed no severe adverse outcomes. Conclusions:. Using point-of-care fully automated flow cytometry to analyze the neutrophil compartment in geriatric hip fracture patients is feasible and holds clinical value in determining patients at risk for adverse outcome. This study is a first step toward immuno-based precision medicine for identifying geriatric hip fracture patients that are deemed fit for surgery.

IntroductionExtensive trauma surgery evokes an immediate cellular immune response including altered circulatory neutrophil numbers. The concurrent bone marrow (BM) response however is currently unclear. We hypothesize that these BM changes include (1) a relative reduction of the bone marrow neutrophil fraction and (2) increasing heterogeneity of the bone marrow neutrophil pool due to (3) the appearance of aged/returning neutrophils from circulation into the BM-compartment.Materials and MethodsEight pigs were included in a standardized extensive trauma surgery model. Blood and bone marrow samples were collected at baseline and after 3 hours of ongoing trauma surgery. Leukocyte and subtype counts and cell surface receptor expression levels were studied by flow cytometry.ResultsAll animals survived the interventions. A significant drop in circulating neutrophil counts from 9.3 to 3.2x106 cells/ml (P=0.001) occurred after intervention, whereas circulatory neutrophil cell surface expression of CD11b increased. The concurrent bone marrow response included an increase of the BM neutrophil fraction from 63 ± 3 to 71 ± 3 percent (P

Abstract Background Patients often develop infectious complications after severe trauma. No biomarkers exist that enable early identification of patients who are at risk. Neutrophils are important immune cells that combat these infections by phagocytosis and killing of pathogens. Analysis of neutrophil function used to be laborious and was therefore not applicable in routine diagnostics. Hence, we developed a quick and point-of-care method to assess a critical part of neutrophil function, neutrophil phagosomal acidification. The aim of this study was to investigate whether this method was able to analyze neutrophil functionality in severely injured patients and whether a relation with the development of infectious complications was present. Results Fifteen severely injured patients (median ISS of 33) were included, of whom 6 developed an infection between day 4 and day 9 after trauma. The injury severity score did not significantly differ between patients who developed an infection and patients who did not (p = 0.529). Patients who developed an infection showed increased acidification immediately after trauma (p = 0.006) and after 3 days (p = 0.026) and a decrease in the days thereafter to levels in the lower normal range. In contrast, patients who did not develop infectious complications showed high-normal acidification within the first days and increased tasset to identify patients at risk for infections after trauma and to monitor the inflammatory state of these trauma patients. Conclusion Neutrophil function can be measured in the ICU setting by rapid point-of-care analysis of phagosomal acidification. This analysis differed between trauma patients who developed infectious complications and trauma patients who did not. Therefore, this assay might prove a valuable asset to identify patients at risk for infections after trauma and to monitor the inflammatory state of these trauma patients. Trial registration Central Committee on Research Involving Human Subjects, NL43279.041.13. Registered 14 February 2014. https://www.toetsingonline.nl/to/ccmo_search.nsf/Searchform?OpenForm .

IntroductionThere is an urgent medical need to differentiate active tuberculosis (ATB) from latent tuberculosis infection (LTBI) and prevent undertreatment and overtreatment. The aim of this study was to identify biomarker profiles that may support the differentiation between ATB and LTBI and to validate these signatures.Materials and MethodsThe discovery cohort included adult individuals classified in four groups: ATB (n = 20), LTBI without prophylaxis (untreated LTBI; n = 20), LTBI after completion of prophylaxis (treated LTBI; n = 20), and healthy controls (HC; n = 20). Their sera were analyzed for 40 cytokines/chemokines and activity of adenosine deaminase (ADA) isozymes. A prediction model was designed to differentiate ATB from untreated LTBI using sparse partial least squares (sPLS) and logistic regression analyses. Serum samples of two independent cohorts (national and international) were used for validation.ResultssPLS regression analyses identified C-C motif chemokine ligand 1 (CCL1), C-reactive protein (CRP), C-X-C motif chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) as the most discriminating biomarkers. These markers and ADA(2) activity were significantly increased in ATB compared to untreated LTBI (p ≤ 0.007). Combining CCL1, CXCL10, VEGF, and ADA2 activity yielded a sensitivity and specificity of 95% and 90%, respectively, in differentiating ATB from untreated LTBI. These findings were confirmed in the validation cohort including remotely acquired untreated LTBI participants.ConclusionThe biomarker signature of CCL1, CXCL10, VEGF, and ADA2 activity provides a promising tool for differentiating patients with ATB from non-treated LTBI individuals.

Summary: Mepolizumab (anti-IL-5) is a successful biological for treatment of T2/eosinophilic asthma by blocking the IL-5-eosinophil axis. The kinetics of human eosinophils in blood and sputum was determined to better understand the underlying mechanism(s). Pulse-chase labeling was performed with 6,6-2H2-glucose in patients with asthma after short term (4 days) and long term (84 days) treatment with mepolizumab (n = 10) or placebo (n = 10). The retention time of eosinophils in sputum was longer than in blood. Treatment with mepolizumab induced a fast and long-lasting eosinopenia with no reduction of eosinophil progenitors. The retention time of eosinophils in blood was delayed only after short-term treatment. This leads to the hypothesis that IL-5 increases the number of IL-5-responsive progenitors and potentiates homing to the tissues, leading to reactive eosinophilia. Long-term treatment is associated with low numbers of IL-5-independent eosinophils in blood and tissues. Therefore, long-term treatment with mepolizumab restores the kinetics of eosinophils as normally found in homeostasis.

At homeostasis the vast majority of neutrophils in the circulation expresses CD16 and CD62L within a narrow expression range, but this quickly changes in disease. Little is known regarding the changes in kinetics of neutrophils phenotypes in inflammatory conditions. During acute inflammation more heterogeneity was found, characterized by an increase in CD16dim banded neutrophils. These cells were probably released from the bone marrow (left shift). Acute inflammation induced by human experimental endotoxemia (LPS model) was additionally accompanied by an immediate increase in a CD62Llow neutrophil population, which was not as explicit after injury/trauma induced acute inflammation. The situation in sub-acute inflammation was more complex. CD62Llow neutrophils appeared in the peripheral blood several days (>3 days) after trauma with a peak after 10 days. A similar situation was found in the blood of COVID-19 patients returning from the ICU. Sorted CD16low and CD62Llow subsets from trauma and COVID-19 patients displayed the same nuclear characteristics as found after experimental endotoxemia. In diseases associated with chronic inflammation (stable COPD and treatment naive HIV) no increases in CD16low or CD62Llow neutrophils were found in the peripheral blood. All neutrophil subsets were present in the bone marrow during homeostasis. After LPS rechallenge, these subsets failed to appear in the circulation, but continued to be present in the bone marrow, suggesting the absence of recruitment signals. Because the subsets were reported to have different functionalities, these results on the kinetics of neutrophil subsets in a range of inflammatory conditions contribute to our understanding on the role of neutrophils in health and disease.

Background: Coronavirus disease of 2019 (COVID-19) is associated with a prothrombotic state and a high incidence of thrombotic event(s) (TE).Objectives: To study platelet reactivity in hospitalized COVID-19 patients and determine a possible association with the clinical outcomes thrombosis and all-cause mortality.Methods: Seventy nine hospitalized COVID-19 patients were enrolled in this retrospective cohort study and provided blood samples in which platelet reactivity in response to stimulation with ADP and TRAP-6 was determined using flow cytometry. Clinical outcomes included thrombotic events, and all-cause mortality.Results: The incidence of TE in this study was 28% and all-cause mortality 16%. Patients that developed a TE were younger than patients that did not develop a TE [median age of 55 vs. 70 years; adjusted odds ratio (AOR) = 0.96 per 1 year of age, 95% confidence interval (CI) 0.92–1.00; p = 0.041]. Furthermore, patients using preexisting thromboprophylaxis were less likely to develop a thrombotic complication than patients that were not (18 vs. 54%; AOR = 0.19, 95% CI 0.04–0.84; p = 0.029). Conversely, having asthma strongly increased the risk on TE development (AOR = 6.2, 95% CI 1.15–33.7; p = 0.034). No significant differences in baseline P-selectin expression or platelet reactivity were observed between the COVID-19 positive patients (n = 79) and COVID-19 negative hospitalized control patients (n = 21), nor between COVID-19 positive survivors or non-survivors. However, patients showed decreased platelet reactivity in response to TRAP-6 following TE development.Conclusion: We observed an association between the use of preexisting thromboprophylaxis and a decreased risk of TE during COVID-19. This suggests that these therapies are beneficial for coping with COVID-19 associated hypercoagulability. This highlights the importance of patient therapy adherence. We observed lowered platelet reactivity after the development of TE, which might be attributed to platelet desensitization during thromboinflammation.

Abstract Background Trauma leads to a complex inflammatory cascade that induces both immune activation and a refractory immune state in parallel. Although both components are deemed necessary for recovery, the balance is tight and easily lost. Losing the balance can lead to life-threatening infectious complications as well as long-term immunosuppression with recurrent infections. Neutrophils are known to play a key role in these processes. Therefore, this review focuses on neutrophil characteristics and function after trauma and how these features can be used to identify trauma patients at risk for infectious complications. Results Distinct neutrophil subtypes exist that play their own role in the recovery and/or development of infectious complications after trauma. Furthermore, the refractory immune state is related to the risk of infectious complications. These findings change the initial concepts of the immune response after trauma and give rise to new biomarkers for monitoring and predicting inflammatory complications in severely injured patients. Conclusion For early recognition of patients at risk, the immune system should be monitored. Several neutrophil biomarkers show promising results and analysis of these markers has become accessible to such extent that they can be used for point-of-care decision making after trauma.

Leukocyte viability (determined by e.g. propidium iodide [PI] staining) is automatically measured by hematology analyzers to check for delayed bench time. Incidental findings in fresh blood samples revealed the existence of leukocytes with decreased viability in critically ill surgical patients. Not much is known about these cells and their functional and/or clinical implications. Therefore, we investigated the incidence of decreased leukocyte viability, the implications for leukocyte functioning and its relation with clinical outcomes. An automated alarm was set in a routine hematology analyzer (Cell-Dyn Sapphire) for the presence of non-viable leukocytes characterized by increased fluorescence in the PI-channel (FL3:630±30nm). Patients with non-viable leukocytes were prospectively included and blood samples were drawn to investigate leukocyte viability in detail and to investigate leukocyte functioning (phagocytosis and responsiveness to a bacterial stimulus). Then, a retrospective analysis was conducted to investigate the incidence of fragile neutrophils in the circulation and clinical outcomes of surgical patients with fragile neutrophils hospitalized between 2013-2017. A high FL3 signal was either caused by 1) neutrophil autofluorescence which was considered false positive, or by 2) actual non-viable PI-positive neutrophils in the blood sample. These two causes could be distinguished using automatically generated data from the hematology analyzer. The non-viable (PI-positive) neutrophils proved to be viable (PI-negative) in non-lysed blood samples, and were therefore referred to as 'fragile neutrophils'. Overall leukocyte functioning was not impaired in patients with fragile neutrophils. Of the 11 872 retrospectively included surgical patients, 75 (0.63%) were identified to have fragile neutrophils during hospitalization. Of all patients with fragile neutrophils, 75.7% developed an infection, 70.3% were admitted to the ICU and 31.3% died during hospitalization. In conclusion, fragile neutrophils occur in the circulation of critically ill surgical patients. These cells can be automatically detected during routine blood analyses and are an indicator of critical illness.

Background and purpose—Nonunion is common in femoral fractures. Previous studies suggested that the systemic immune response after trauma can interfere with fracture healing. Therefore, we investigated whether there is a relation between peripheral blood cell counts and healing of femur fractures. Patients and methods—62 multi-trauma patients with a femoral fracture presenting at the University Medical Centre Utrecht between 2007 and 2013 were retrospectively included. Peripheral blood cell counts from hematological analyzers were recorded from the 1st through the 14th day of the hospital stay. Generalized estimating equations were used to compare outcome groups. Results—12 of the 62 patients developed nonunion of the femoral fracture. The peripheral blood-count curves of total leukocytes, neutrophils, monocytes, lymphocytes, and platelets were all statistically significantly lower in patients with nonunion, coinciding with significantly higher CRP levels during the first 2 weeks after trauma in these patients. Interpretation—Patients who developed femoral nonunion after major trauma demonstrated lower numbers of myeloid cells in the peripheral blood than patients with normal fracture healing. This absent rise of myeloid cells seems to be related to a more severe post-traumatic immune response.

Abstract: Neutrophils comprise a heterogeneous population of cells essential for bacterial eradication, and defects in neutrophil function are associated with increased susceptibility to infection. In this study, neutrophils from healthy controls were shown to prevent bacterial proliferation for at least 48 hours when cocultured with methicillin-resistant Staphylococcus aureus (MRSA) in tissue-like scaffolds by establishing a bacteriostatic environment inside their phagolysosome. This intracellular bacterial containment is independent of reactive oxygen species because neutrophils that lack a functional nicotinamide adenine dinucleotide phosphate–oxidase complex displayed no defect in intracellular bacterial containment, whereas killing of the pathogen was impaired. During acute inflammation, a subset of CD16bright/CD62Ldim hypersegmented neutrophils displayed normal phagocytosis associated with a remarkably poor capacity to contain bacteria intracellularly. Conversely, CD16dim-banded neutrophils were the only neutrophil subset that adequately contained MRSA. These findings demonstrate a clear neutrophil heterogeneity in their antimicrobial capacity and the appearance of neutrophil subsets with a clear differentiation in functionality during acute inflammation. Furthermore, this study provides an evolutionary basis for the rapid release of banded neutrophils into the circulation during acute inflammation.

A malfunction of the innate immune response in COVID-19 is associated with eosinopenia, particularly in more severe cases. This study tested the hypothesis that this eosinopenia is COVID-19 specific and is associated with systemic activation of eosinophils. Blood of 15 healthy controls and 75 adult patients with suspected COVID-19 at the ER were included before PCR testing and analyzed by point-of-care automated flow cytometry (CD10, CD11b, CD16, and CD62L) in the absence or presence of a formyl peptide (fNLF). Forty-five SARS-CoV-2 PCR positive patients were grouped based on disease severity. PCR negative patients with proven bacterial (n = 20) or other viral (n = 10) infections were used as disease controls. Eosinophils were identified with the use of the FlowSOM algorithm. Low blood eosinophil numbers (p < 0.005) were found both in patients with COVID-19 and with other infectious diseases, albeit less pronounced. Two discrete eosinophil populations were identified in healthy controls both before and after activation with fNLF based on the expression of CD11b. Before activation, the CD11bbright population consisted of 5.4% (CI95% = 3.8, 13.4) of total eosinophils. After activation, this population of CD11bbright cells comprised nearly half the population (42.21%, CI95% = 35.9, 54.1). Eosinophils in COVID-19 had a similar percentage of CD11bbright cells before activation (7.6%, CI95% = 4.5, 13.6), but were clearly refractory to activation with fNLF as a much lower percentage of cells end up in the CD11bbright fraction after activation (23.7%, CI95% = 18.5, 27.6; p < 0.001). Low eosinophil numbers in COVID-19 are associated with refractoriness in responsiveness to fNLF. This might be caused by migration of fully functional cells to the tissue.

Abstract Multicolour Flow Cytometry (MFC) produces multidimensional analytical data on the quantitative expression of multiple markers on single cells. This data contains invaluable biomedical information on (1) the marker expressions per cell, (2) the variation in such expression across cells, (3) the variability of cell marker expression across samples that (4) may vary systematically between cells collected from donors and patients. Current conventional and even advanced data analysis methods for MFC data explore only a subset of these levels. The Discriminant Analysis of MultiAspect CYtometry (DAMACY) we present here provides a comprehensive view on health and disease responses by integrating all four levels. We validate DAMACY by using three distinct datasets: in vivo response of neutrophils evoked by systemic endotoxin challenge, the clonal response of leukocytes in bone marrow of acute myeloid leukaemia (AML) patients, and the complex immune response in blood of asthmatics. DAMACY provided good accuracy 91–100% in the discrimination between health and disease, on par with literature values. Additionally, the method provides figures that give insight into the marker expression and cell variability for more in-depth interpretation, that can benefit both physicians and biomedical researchers to better diagnose and monitor diseases that are reflected by changes in blood leukocytes.

Abstract Parametric response mapping (PRM) of paired CT lung images has been shown to improve the phenotyping of COPD by allowing for the visualization and quantification of non-emphysematous air trapping component, referred to as functional small airways disease (fSAD). Although promising, large variability in the standard method for analyzing PRMfSAD has been observed. We postulate that representing the 3D PRMfSAD data as a single scalar quantity (relative volume of PRMfSAD) oversimplifies the original 3D data, limiting its potential to detect the subtle progression of COPD as well as varying subtypes. In this study, we propose a new approach to analyze PRM. Based on topological techniques, we generate 3D maps of local topological features from 3D PRMfSAD classification maps. We found that the surface area of fSAD (SfSAD) was the most robust and significant independent indicator of clinically meaningful measures of COPD. We also confirmed by micro-CT of human lung specimens that structural differences are associated with unique SfSAD patterns, and demonstrated longitudinal feature alterations occurred with worsening pulmonary function independent of an increase in disease extent. These findings suggest that our technique captures additional COPD characteristics, which may provide important opportunities for improved diagnosis of COPD patients.

Abstract Background COPD is a heterogeneous chronic inflammatory disease of the airways and it is well accepted that the GOLD classification does not fully represent the complex clinical manifestations of COPD and this classification therefore is not well suited for phenotyping of individual patients with COPD. Besides the chronic inflammation in the lung compartment, there is also a systemic inflammation present in COPD patients. This systemic inflammation is associated with elevated levels of cytokines in the peripheral blood, but the precise composition is unknown. Therefore, differences in phenotype of peripheral blood neutrophils in vivo could be used as a read out for the overall systemic inflammation in COPD. Method Our aim was to utilize an unsupervised method to assess the proteomic profile of peripheral neutrophils of stable COPD patients and healthy age matched controls to find potential differences in these profiles as read-out of inflammatory phenotypes. We performed fluorescence two-dimensional difference gel electrophoresis with the lysates of peripheral neutrophils of controls and stable COPD patients. Results We identified two groups of COPD patients based on the differentially regulated proteins and hierarchical clustering whereas there was no difference in lung function between these two COPD groups. The neutrophils from one of the COPD groups were less responsive to bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). Conclusion This illustrates that systemic inflammatory signals do not necessarily correlate with the GOLD classification and that inflammatory phenotyping can significantly add in an improved diagnosis of single COPD patients. Trial registration Clinicaltrials.gov: NCT00807469 registered December 11th 2008

Neutrophils have been extensively described in the pathophysiology of autoimmune and infectious diseases. Accumulating evidence also suggests the important role of neutrophils in cancer progression through their interaction with cancer and immune cells in blood and in the tumor microenvironment (TME). Most studies have described neutrophils as key drivers of cancer progression, due to their involvement in various tumor promoting functions including proliferation, aggressiveness, and dissemination, as well as in immune suppression. However, such studies were focusing on late-stages of tumorigenesis, in which chronic inflammation had already developed. The role of tumor-associated neutrophils (TANs) at early stages of tumor development remains poorly described, though recent findings indicate that early-stage TANs may display anti-tumor properties. Beyond their role at tumor site, evidence supported by NLR retrospective studies and functional analyses suggest that blood neutrophils could also actively contribute to tumorigenesis. Hence, it appears that the phenotype and functions of neutrophils vary greatly during tumor progression, highlighting their heterogeneity. The origin of pro- or anti-tumor neutrophils is generally believed to arise following a change in cell state, from resting to activated. Moreover, the fate of neutrophils may also involve distinct differentiation programs yielding various subsets of pro or anti-tumor neutrophils. In this review, we will discuss the current knowledge on neutrophils heterogeneity across different tissues and their impact on tumorigenesis, as well as neutrophil-based therapeutic strategies that have shown promising results in pre-clinical studies, paving the way for the design of neutrophil-based next generation immunotherapy.

Receptors recognizing the Fc-part of immunoglobulins (FcR) are important in the engagement of phagocytes with opsonized micro-organisms, but they also play a major role in the pathogenesis of chronic inflammatory diseases. Different FcRs are specifically recognizing and binding the different classes of immunoglobulins, transmitting different signals into the cell. The function of IgG (FcγR's) and IgA (FcαR) recognizing receptors is controlled by cellular signals evoked by activation of heterologous receptors in a process generally referred to as inside-out control. This concept is clearly described for the regulation of integrin receptors. Inside-out control can be achieved at different levels by modulation of: (i) receptor affinity, (ii) receptor avidity/valency, (iii) interaction with signaling chains, (iv) interaction with other receptors and (v) localization in functionally different membrane domains. The inside-out control of FcRs is an interesting target for novel therapy by therapeutical antibodies as it can potentiate or decrease the functionality of the response to the antibodies depending on the mechanisms of the diseases they are applied for.

IgA binding to FcαRI (CD89) is rapidly enhanced by cytokine induced inside-out signaling. Dephosphorylation of serine 263 in the intracellular tail of FcαRI by PP2A and PI3K activation are instrumental in this process. To further investigate these signaling pathways, we targeted downstream kinases of PI3K. Our experiments revealed that PI3K activates PKCζ, which subsequently inhibits GSK-3, a constitutively active kinase in resting cells and found here to be associated with FcαRI. We propose that GSK-3 maintains FcαRI in an inactive state at homeostatic conditions. Upon cytokine stimulation, GSK-3 is inactivated through a PI3K-PKCζ pathway, preventing the maintenance of phosphorylated inactive FcαRI. The concomitantly activated PP2A is then able to dephosphorylate and activate FcαRI. Moreover, FRAP and FLIP studies showed that FcαRI activation coincides with an increased mobile fraction of the receptor. This can enhance FcαRI valency and contribute to stronger avidity for IgA immune complexes. This tightly regulated inside-out signaling pathway allows leukocytes to respond rapidly and efficiently to their environment and could be exploited to enhance the efficacy of future IgA therapeutics.

Neutrophils are main players in the effector phase of the host defense against micro-organisms and have a major role in the innate immune response. Neutrophils show phenotypic heterogeneity and functional flexibility, which highlight their importance in regulation of immune function. However, neutrophils can play a dual role and besides their antimicrobial function, deregulation of neutrophils and their hyperactivity can lead to tissue damage in severe inflammation or trauma. Neutrophils also have an important role in the modulation of the immune system in response to severe injury and trauma. In this review we will provide an overview of the current understanding of neutrophil subpopulations and their function during and post-infection and discuss the possible mechanisms of immune modulation by neutrophils in severe inflammation.

Nowadays, more trauma patients develop chronic critical illness (CCI), a state characterized by prolonged intensive care. Some of these CCI patients have disproportional difficulties to recover and suffer from recurrent infections, a syndrome described as the persistent inflammation, immunosuppression and catabolism syndrome (PICS). A total of 78 trauma patients with an ICU stay of ≥14 days (CCI patients) between 2007 and 2017 were retrospectively included. Within this group, PICS patients were identified through two ways: (1) their clinical course (≥3 infectious complications) and (2) by laboratory markers suggested in the literature (C-reactive protein (CRP) and lymphocytes), both in combination with evidence of increased catabolism. The incidence of PICS was 4.7 per 1000 multitrauma patients. The sensitivity and specificity of the laboratory markers was 44% and 73%, respectively. PICS patients had a longer hospital stay (median 83 vs. 40, p < 0.001) and required significantly more surgical interventions (median 13 vs. 3, p = 0.003) than other CCI patients. Thirteen PICS patients developed sepsis (72%) and 12 (67%) were readmitted at least once due to an infection. In conclusion, patients who develop PICS experience recurrent infectious complications that lead to prolonged hospitalization, many surgical procedures and frequent readmissions. Therefore, PICS forms a substantial burden on the patient and the hospital, despite its low incidence.

Metaplasia in Barrett’s esophagus (BE) is characterized by the transition of squamous epithelium into intestinal-type columnar epithelium. The immune response in BE shares many similarities with the response found in the gut, which is different from the response found in a normal-looking esophagus. Here, we investigated the role of the genes associated with the retinoic acid (RA) pathway in BE, as RA is important not only in shaping the gut’s immune response but also in the induction of metaplasia in vitro. mRNA was isolated from esophageal and duodenal biopsies from BE (n = 14), reflux esophagitis patients (n = 9), and controls (n = 12). cDNA was made and qPCR was performed. The expression of RALDH1, CYP26A1, MAdCAM1 were similar for both the BE and duodenum, but different when compared to squamous esophageal epithelium. BE was characterized by a higher expression of RALDH2 and FOXP3, compared to the duodenum. In BE, RALDH2 correlated with expression of the myeloid dendritic cell-specific genes: CD11c and CD1c. Also, RALDH2 expression correlated with RAR-β and FOXP3. Hierarchical clustering on the expression of multiple relevant genes demonstrated that BE, duodenum, and SQ tissues are clustered as three different groups. The differential expression of RA-specific genes and dendritic cell (DC)-subsets indicates that BE resembles duodenal tissue. The higher expression of RALDH2 and FOXP3 in BE points at a mechanism associated with a possible anti-inflammatory microenvironment. This aberrant immune regulation might contribute to the altered tissue and immune responses found in BE.

Although controlled local inflammation is essential for adequate bone regeneration, several studies have shown that hyper-inflammatory conditions after major trauma are associated with impaired fracture healing. These hyper-inflammatory conditions include the trauma-induced systemic inflammatory response to major injury, open fractures, and significant injury to the surrounding soft tissues. The current literature suggests that increased or prolonged influx of neutrophils into the fracture hematoma may mediate impairment of bone regeneration after hyper-inflammatory conditions. The underlying mechanism remains unclear. We hypothesize that high neutrophil numbers inhibit synthesis of mineralized extracellular matrix (ECM) by bone marrow stromal cells (BMSCs). We therefore studied the effect of increasing concentrations of neutrophils on ECM synthesis by human BMSCs in vitro. Moreover, we determined how high neutrophil concentrations affect BMSC cell counts, as well as BMSC osteogenic activity determined by alkaline phosphatase (ALP) expression and ALP activity. Co-culture of BMSCs with neutrophils induced a 52% decrease in BMSC cell count (p

Endurance exercise is associated with a transient increase in neutrophil counts in the peripheral blood. Here we investigate the impact of intensified endurance exercise on the neutrophil compartment. We hypothesized that intensified endurance exercise leads to mobilization of neutrophil subsets, which are normally absent in the blood. Furthermore, we followed the potential build-up of neutrophil activation and the impact on overnight recovery of the neutrophil compartment during a seven-day cycling tour. The neutrophil compartment was studied in 28 healthy amateur cyclists participating in an eight-day strenuous cycling tour. Blood samples were taken at baseline, after 4 days and after 7 days of cycling. The neutrophil compartment was analyzed in terms of numbers and its phenotype by deep phenotyping of flow cytometry data with the multi-dimensional analysis method FLOOD. Repeated endurance exercise led to a gradual increase in total neutrophil counts over the days leading to a 1.26 fold-increase (95%CI 1.01-1.51 p = 0.0431) in the morning of day 8. Flow cytometric measurements revealed the appearance of 2 additional neutrophil subsets: CD16brightCD62Ldim and CD16dimCD62Lbright. A complex change in neutrophil phenotypes was present characterized by decreased expression of both CD11b and CD62L and marked increased expression of LAIR-1, VLA-4 and CBRM1/5. The changes in expression were found on all neutrophils present in the blood. Strikingly, in strong contrast to our findings during acute inflammation evoked by LPS challenge, these neutrophils did not upregulate classical degranulation markers. In fact, our FLOOD analysis revealed that the exercise induced neutrophil phenotype did not overlap with the neutrophil subsets arising upon acute inflammation. In conclusion, during multiple days of endurance exercise the neutrophil compartment does not regain homeostasis overnight. Thereby our study supports the concept of a build-up of inflammatory cues during repeated endurance exercise training, causing a prolonged change of the systemic neutrophil compartment.

IntroductionPhagocytosis by neutrophils is a key process in the innate immune response against invading microorganisms. Despite reported heterogeneity in other neutrophils functions, little is known regarding differences in phagocytosis by individual cells. Therefore, we tested the hypothesis that heterogeneity is present in the neutrophil compartment in its potency to phagocytize bacteria.MethodsPhagocytosis assays were performed in suspension with isolated neutrophils and Staphylococcus aureus expressing different fluorescent proteins at MOIs between 1 and 10. Repetitive addition of bacteria with different fluorescent proteins and MOIs was used to compare the phagocytic capacity of S. aureus-green fluorescent protein (GFP)-positive and negative neutrophils and exclude randomness.ResultsThe percentage and mean fluorescence intensity (MFI) of S. aureus-GFP-positive neutrophils increased with higher MOIs. The increase in MFI was due to phagocytosis of multiple bacteria per neutrophil as was confirmed by confocal imaging. Sequential phagocytosis of GFP- and mCherry-expressing S. aureus showed a non-random process, as S. aureus-GFP-positive neutrophils preferentially phagocytized S. aureus-mCherry.ConclusionAll neutrophils were able to phagocytize S. aureus, but some were much more potent than others. Therefore, at physiologically relevant MOIs these potent phagocytizing neutrophils will outcompete the uptake of bacteria by less competent cells in a process we propose to name “competitive phagocytosis.”

Severe trauma can lead to the development of infectious complications after several days, such as sepsis. Early identification of patients at risk will aid anticipating these complications. The aim of this study was to test the relation between the acute (5 days) development of septic complications and validate this in different trauma populations.Two prospective, observational, cohort series in the Netherlands and South Africa, consisting of severely injured trauma patients. Neutrophil responsiveness by fMLF-induced active FcγRII was measured in whole blood flowcytometry, as read out for the systemic immune response within hours after trauma. Sepsis was scored daily. Ten of the 36 included Dutch patients developed septic shock. In patients with septic shock, neutrophils showed a lower expression of fMLF-induced active FcγRII immediately after trauma when compared to patients without septic shock (P = 0.001). In South Africa 11 of 73 included patients developed septic shock. Again neutrophils showed lower expression of fMLF induced active FcγRII (P = 0.001). In the combined cohort, all patients who developed septic shock demonstrated a decreased neutrophil responsiveness.Low responsiveness of neutrophils for the innate stimulus fMLF immediately after trauma preceded the development of septic shock during admission by almost a week and did not depend on a geographical/racial background, hospital protocols and health care facilities. Decreased neutrophil responsiveness appears to be a prerequisite for septic shock after trauma. This might enable anticipation of this severe complication in trauma patients.

Neutrophils are important assets in defense against invading bacteria like staphylococci. However, (dysfunctioning) neutrophils can also serve as reservoir for pathogens that are able to survive inside the cellular environment. Staphylococcus aureus is a notorious facultative intracellular pathogen. Most vulnerable for neutrophil dysfunction and intracellular infection are immune-deficient patients or, as has recently been described, severely injured patients. These dysfunctional neutrophils can become hide-out spots or “Trojan horses” for S. aureus. This location offers protection to bacteria from most antibiotics and allows transportation of bacteria throughout the body inside moving neutrophils. When neutrophils die, these bacteria are released at different locations. In this review, we therefore focus on the capacity of several groups of antibiotics to enter human neutrophils, kill intracellular S. aureus and affect neutrophil function. We provide an overview of intracellular capacity of available antibiotics to aid in clinical decision making. In conclusion, quinolones, rifamycins and sulfamethoxazole-trimethoprim seem very effective against intracellular S. aureus in human neutrophils. Oxazolidinones, macrolides and lincosamides also exert intracellular antibiotic activity. Despite that the reviewed data are predominantly of in vitro origin, these findings should be taken into account when intracellular infection is suspected, as can be the case in severely injured patients.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation caused by ongoing inflammatory and remodeling processes of the airways and lung tissue. Inflammation can be targeted by corticosteroids. However, airway inflammation is generally less responsive to steroids in COPD than in asthma. The underlying mechanisms are yet unclear. This study aimed to assess whether skin corticosteroid insensitivity is associated with COPD and COPD severity using the corticosteroid skin blanching test.COPD patients GOLD stage I-IV (n = 27, 24, 22, and 16 respectively) and healthy never-smokers and smokers (n = 28 and 56 respectively) were included. Corticosteroid sensitivity was assessed by the corticosteroid skin blanching test. Budesonide was applied in 8 logarithmically increasing concentrations (0-100 μg/ml) on subject's forearm. Assessment of blanching was performed after 7 hours using a 7-point scale (normal skin to intense blanching). All subjects performed spirometry and body plethysmography.Both GOLD III and GOLD IV COPD patients showed significantly lower skin blanching responses than healthy never-smokers and smokers, GOLD I, and GOLD II patients. Their area under the dose-response curve values of the skin blanching response were 586 and 243 vs. 1560, 1154, 1380, and 1309 respectively, p

BACKGROUND AND OBJECTIVE:Reflux esophagitis (RE) is characterized by inflammation of the squamous epithelium (SQ) of the esophagus and may progress to Barrett's esophagus (BE) characterized by intestinal metaplasia. The role of inflammation in this transition has been postulated but lacks experimental evidence. Here, the inflammatory responses in the esophagus of these patients were investigated. PATIENTS AND METHODS:Fifty-one esophageal biopsies from with patients BE (n = 19), RE (n = 8) and controls (n = 23) were analyzed. T-cells were analyzed before and after ex vivo expansion (14 days) by multicolor flow cytometric analysis. The following markers were studied: CD3, CD4, CD8 (T-cell markers), Granzyme B (marker of cytotoxicity), CD103 (αE/epithelial integrin) and NKg2a (inhibitory receptor on T-cells and NK-cells). RESULTS:Analysis of ex vivo cultures from normal looking SQ from controls, RE patients, and BE patients revealed no significant differences in the number and phenotypes of T-cells. In contrast, tissue from RE was different to normal SQ in four aspects: 1) higher percentages of CD3+ CD4+-cells (72±7% vs 48±6%, p = 0.01) and 2) CD8+ GranzymeB+-cells (53±11% vs 26±4%, p

Leptin is an adipokine that is thought to be important in many inflammatory diseases, and is known to influence the function of several leukocyte types. However, no clear consensus is present regarding the responsiveness of neutrophils for this adipokine. In this study a 2D DIGE proteomics approach was used as an unbiased approach to identify leptin-induced effects on neutrophils. Additionally chemotaxis and survival experiments were performed to reproduce results from literature showing putative effects of leptin on these neutrophil responses. Leptin did not induce any significant changes in the proteome provided leptin was added at physiologically relevant concentrations (250 ng). Our leptin batches were biologically active as they induced proliferation in LeptinR expressing Ba/F3 cells. At high concentrations (25000 ng) leptin induced a change in neutrophil proteome. Seventeen differently regulated spots were identified of which twelve could be characterized by mass spectrometry. Two of these identified proteins, SerpinB1 and p40 phox, were chosen for further analysis but leptin-induced expression analyzed by western blot were highly variable. Additionally leptin also induced neutrophil survival at these high concentrations. No leptin-induced chemotaxis of human neutrophils was detected at any concentration. In conclusion, physiological concentrations of leptin do not affect neutrophils. High leptin concentrations induced survival and changes in the neutrophils proteome, but this was most likely mediated by an indirect effect. However, it cannot be ruled out that the effects were mediated by a yet not-identified leptin receptor on human neutrophils.

During systemic inflammation different neutrophil subsets are mobilized to the peripheral blood. These neutrophil subsets can be distinguished from normal circulating neutrophils (CD16(bright)/CD62L(bright)), based on either an immature CD16(dim)/CD62L(bright) or a CD16(bright)/CD62L(dim) phenotype. Interestingly, the latter neutrophil subset is known to suppress lymphocyte proliferation ex vivo, but how neutrophils become suppressive is unknown. We performed transcriptome analysis on the different neutrophil subsets to identify changes in mRNA expression that are relevant for their functions. Neutrophil subsets were isolated by fluorescence-activated cell sorting from blood of healthy volunteers that were administered a single dose of lipopolysaccharide (2 ng/kg i.v.) and the transcriptome was determined by microarray analysis. Interestingly, the CD16(bright)/CD62L(dim) suppressive neutrophils showed an interferon-induced transcriptome profile. More importantly, IFN-γ, but not IFN-α or IFN-β stimulated neutrophils, acquired the capacity to suppress lymphocyte proliferation through the expression of programmed death ligand 1 (PD-L1). These data demonstrate that IFN-γ-induced expression of PD-L1 on neutrophils enables suppression of lymphocyte proliferation. Specific stimulation of neutrophils present at the inflammatory sites might therefore have a pivotal role in regulating lymphocyte-mediated inflammation and autoimmune disease.

Cigarette smoking induces inflammatory responses in all smokers and is the major risk factor for lung disease such as chronic obstructive pulmonary disease (COPD). In this progressive disease, chronic inflammation in the lung contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). The generation of this tripeptide is mediated by a multistep pathway involving matrix metalloproteases (MMPs) 8 and 9 and prolyl endopeptidase (PE). Here we investigated whether cigarette smoke extract (CSE) stimulates human PMNs to breakdown whole matrix collagen leading to the generation of the chemotactic collagen fragment N-ac-PGP.Incubating PMNs with CSE led to the release of chemo-attractant CXCL8 and proteases MMP8 and MMP9. PMNs constitutively expressed PE activity as well as PE protein. Incubating CSE-primed PMNs with collagen resulted in collagen breakdown and in N-ac-PGP generation. Incubation of PMNs with the tripeptide N-ac-PGP resulted in the release of CXCL8, MMP8 and MMP9. Moreover, we tested whether PMNs from COPD patients are different from PMNs from healthy donors. Here we show that the intracellular basal PE activity of PMNs from COPD patients increased 25-fold compared to PMNs from healthy donors. Immunohistological staining of human lung tissue for PE showed that besides neutrophils, macrophages and epithelial cells express PE.This study indicates that neutrophils activated by cigarette smoke extract can breakdown collagen into N-ac-PGP and that this collagen fragment itself can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. MMP-, PE- or PGP-inhibitors can serve as an attractive therapeutic target and may open new avenues towards effective treatment of COPD.

Background and objectiveBarrett's esophagus (BE) is characterized by the transition of squamous epithelium into columnar epithelium with intestinal metaplasia. The increased number and types of immune cells in BE have been indicated to be due to a Th2-type inflammatory process. We tested the alternative hypothesis that the abundance of T-cells in BE is caused by a homing mechanism that is found in the duodenum.Patients and methodsBiopsies from BE and duodenal tissue from 30 BE patients and duodenal tissue from 18 controls were characterized by immmunohistochemistry for the presence of T-cells and eosinophils(eos). Ex vivo expanded T-cells were further phenotyped by multicolor analysis using flowcytometry.ResultsThe high percentage of CD4(+)-T cells (69±3% (mean±SEM/n = 17, by flowcytometry)), measured by flowcytometry and immunohistochemistry, and the presence of non-activated eosinophils found in BE by immunohistochemical staining, were not different from that found in duodenal tissue. Expanded lymphocytes from these tissues had a similar phenotype, characterized by a comparable but low percentage of αE(CD103) positive CD4(+)cells (44±5% in BE, 43±4% in duodenum of BE and 34±7% in duodenum of controls) and a similar percentage of granzyme-B(+)CD8(+) cells(44±5% in BE, 33±6% in duodenum of BE and 36±7% in duodenum of controls). In addition, a similar percentage of α4β7(+) T-lymphocytes (63±5% in BE, 58±5% in duodenum of BE and 62±8% in duodenum of controls) was found. Finally, mRNA expression of the ligand for α4β7, MAdCAM-1, was also similar in BE and duodenal tissue. No evidence for a Th2-response was found as almost no IL-4(+)-T-cells were seen.ConclusionThe immune cell composition (lymphocytes and eosinophils) and expression of intestinal adhesion molecule MAdCAM-1 is similar in BE and duodenum. This supports the hypothesis that homing of lymphocytes to BE tissue is mainly caused by intestinal homing signals rather than to an active inflammatory response.

Introduction. Flow cytometry markers have been proposed as useful predictors for the occurrence of posttraumatic inflammatory complications. However, currently the need for a dedicated laboratory and the labour-intensive analytical procedures make these markers less suitable for clinical practice. We tested an approach to overcome these limitations. Material and Methods. Neutrophils of healthy donors were incubated with antibodies commonly used in trauma research: CD11b (MAC-1), L-selectin (CD62L), FcγRIII (CD16), and FcγRII (CD32) in active form (MoPhab A27). Flow cytometric analysis was performed both on a FACSCalibur, a standard flow cytometer, and on a Cell-Dyn Sapphire, a routine haematology analyser. Results. There was a high level of agreement between the two types of analysers, with 41% for FcγRIII, 80% for L-selectin, 98% for CD11b, and even a 100% agreement for active FcγRII. Moreover, analysis on the routine haematology analyser was possible in less than a quarter of the time in comparison to the flow cytometer. Conclusion. Analysis of neutrophil phenotype on the Cell-Dyn Sapphire leads to the same conclusion compared to a standard flow cytometer. The markedly reduced time necessary for analysis and reduced labour intensity constitutes a step forward in implementation of this type of analysis in clinical diagnostics in trauma research.