Neutrophil Granulocyte Function Research Articles

Recovery of the Decreased Phagocytic Function of Peripheral Monocytes and Neutrophil Granulocytes following Cytoreductive Surgery in Advanced Stage Epithelial Ovarian Cancer.

(1) Monocytes and neutrophil granulocytes are the phagocytic cells of the innate immune system, playing a crucial role in recognizing and eliminating tumor-transformed cells. Our objective was to assess the impact of advanced-stage epithelial ovarian cancer (EOC) and cytoreductive surgery on the phagocytic function of peripheral monocytes and neutrophil granulocytes. We aimed to compare the pre- and postoperative phagocytic function of these immune cells in EOC patients with healthy control women. Additionally, we aimed to examine the influence of surgery on phagocytic function by comparing pre- and postoperative samples from patients with benign gynecological tumors. (2) We examined peripheral blood samples from 20 patients with FIGO IIIC stage high-grade serous EOC and 16 patients with benign gynecological tumors as surgical controls, collected before and seven days after tumor removal surgery, and from 14 healthy women. After separation, the cells were incubated with Zymosan-A particles, and the phagocytic index (PI) was assessed using immunofluorescence microscopy. One-way ANOVA, the Kruskal-Wallis H-test, and the paired samples t-test were used for the statistical analysis of the data. A significance level of p < 0.05 was applied. (3) Peripheral monocytes and neutrophils from EOC patients exhibited significantly lower preoperative PI values compared to healthy controls (p < 0.001; p < 0.001, respectively). Following cytoreductive surgery, the PI values of immune cells in EOC patients significantly increased by the 7th postoperative day (p < 0.001; p < 0.001), reaching levels comparable to those of healthy controls (p = 0.700 and p = 0.991). In contrast, there was no significant disparity in the PI values of cells obtained from pre- and postoperative blood samples of surgical controls when compared to healthy women (monocytes: p = 0.361 and p = 0.303; neutrophils: p = 0.150 and p = 0.235). (4) EOC and/or its microenvironment may produce factors that reduce the phagocytic function of monocytes and neutrophils, and the production of these factors may be reduced or eliminated after tumor removal.

The effectiveness of immunomodulatory therapy with a synthetic analogue of the active center of the hormone thymus thymopoietin in the complex treatment of immunocompromised women with chronic infectious and inflammatory diseases of the pelvic organs

Chronic inflammatory diseases of the pelvic organs (pelvic inflammatory disease, PID) in women is among the main understudied problems in gynecology worldwide with adverse medical and socio-economic consequences, thus justifying the need for further study of immunopathogenesis and development of new approaches to treatment. Our objective was to develop new immunotherapeutic approaches to correction of combined disorders of the immune system functioning in immunocompromised women with PID and to evaluate their clinical and immunological efficacy.&#x0D; 55 women aged 20-40 years were examined, i.e., 35 patients with exacerbation of sluggish or recurrent PID, resistant to conventional therapy. Testiung was performed before complex treatment (study group 1 GI- 1) and after the course (study group 2 GI-2). Contents of T and B lymphocytes, natural killer cells (NK) (CYTOMICS FC500, USA), phagocytic and microbicidal functions of neutrophilic granulocytes (NG) were assessed in SG-1 and SG-2 before and 2-3 days after complex treatment with addition of an immunotherapeutic drug based on hexapeptide (HP) at a daily dose of 45 mg/ml intramuscularly for 10 days.&#x0D; In patients from SG-1, a decrease in T cells (CD3+CD19- ) and B cells (CD3-CD19+), a 2-fold increase in the content of NK CD3-CD16+CD56+ was found, along with altered functioning of NG (deficiency of actively phagocytizing NG, a decrease in their digestive function and NADPH-oxidase activity). In SG-2 patients, the treatment was followed by restoration of the T (CD3+CD19-) and B cells (CD3-CD19+), NK cells (CD3-CD16+CD56+), like as an increase in effector functions, i.e., microbial capture by NG and their killing ability due to activation of NADPH oxidases and normalization of microbicidal reserve capacity in the NG cell population. Positive clinical effect included reduction of clinical symptoms in acute period, absence of PID exacerbations over follow-up for 6 months (85.6% of cases). Occasional exacerbations of PID were associated with medical manipulations (5.7%) and unprotected sexual contacts (5.7%).&#x0D; The immunopathogenetically proven approach to correction of combined functional impairment of immune system in the women with PID shows a positive clinical and immunological effect.

Glycopeptide Antibiotics Impair Neutrophil Effector Functions

Introduction: Neutrophilic granulocytes represent the first line of defense against microorganisms. Granulocytes phagocytose microorganisms and specifically synthesize oxygen radicals against them, which eventually kills the invaders. Methods: Neutrophilic granulocytes were isolated from peripheral blood of healthy volunteer donors. Putative interference of new-generation antibiotics with neutrophil function was tested using a collection of granulocyte-stimulating agents and Amplex™ Red-based plate assay and flow cytometry-based respiratory burst assays. In addition, phagocytosis of E. coli, IL-8 production, bactericidal activity, and CD62L expression of granulocytes were evaluated. Results: Of note, we found that the two glycopeptide antibiotics dalbavancin and teicoplanin inhibited ROS production upon granulocyte activation via different signaling pathways in a dose-dependent manner. Dalbavancin also blocked the PMA-induced shedding of CD62L. In contrast, the oxazolidinone antibiotics tedizolid and linezolid had no effect on neutrophil function, while the combination of ceftazidime/avibactam dose dependently inhibited the fMLP/Cytochalasin B-induced granulocyte burst in a dose-dependent manner. Additionally, we showed that dalbavancin and teicoplanin as well as sulfametrole/trimethoprim and ceftazidime/avibactam inhibited baseline and PMA-induced IL-8 production by neutrophilic granulocytes. Moreover, dalbavancin impaired the bactericidal activity of neutrophilic granulocytes. Conclusion: We here identified hitherto unknown inhibitory effects of several classes of antibiotics on the effector functions of neutrophilic granulocytes.

Live bacterial ligands universally regulate mTOR activity but are not essential for homeostatic innate immunity

Abstract Multiple studies have reported significant impact of gut microbiota on hematopoiesis, including granulocyte development and functional defects. Germ-Free (GF) mice show developmental defects due to lack of live bacteria; underscored by the development of Altered Schaedler Flora (ASF), a community of 8 bacterial species sufficient to rescue developmental immune and gastrointestinal defects. Mimicking gnotobiosis by using antibiotics perturbs the microbiome systemically. Therefore, results from such studies may represent sequelae of the drug perturbation rather than simply modifying microbiota. We studied the impact of gut microbiota signals on murine hematopoiesis without antibiotics or fecal transfer. Namely, by using mice (from Taconic) generated by embryonic transfer of C57/Bl6 fertilized eggs into females with controlled microbiota, totaling 4 groups of mice: 1. GF; 2. ASF (no Gammaproteobacteria); 3. Excluded Flora (EF, includes Gammaproteobacteria); 4. WildR 7(wild mouse microbiome). We found no significant differences in either number or effector function of bone-marrow neutrophil granulocytes, myeloid progenitors, or stem cells. The single-cell genomic landscape of CD117+ bone-marrow progenitors confirmed flow analyses but revealed a profound defect in gnotobiotic hematopoietic signatures. Importantly, standard pathogen free C57/BL6 mice were inappropriate controls. While gnotobiotic mice likely receive inert TLR stimulation via bedding and food, the addition of any group of live bacteria was sufficient to rescue gene expression downstream of mTOR signaling. Such robust signatures could be considered so essential to life that they do not depend on any single bacterial clade. This project is supported by NIH R01HL122661.

Immunomodulation of neutrophil granulocyte functions by bacterial polyphosphates.

Polyphosphates are highly conserved, linear polymers of monophosphates that reside in all living cells. Bacteria produce long chains containing hundreds to thousands of phosphate units, which can interfere with host defense to infection. Here, we report that intratracheal long-chain polyphosphate administration to C57BL/6J mice resulted in the release of proinflammatory cytokines and influx of Ly6G+ polymorphonuclear neutrophils in the bronchoalveolar lavage fluid causing a disruption of the physiologic endothelial-epithelial small airway barrier and histologic signs of lung injury. Polyphosphate-induced effects were attenuated after neutrophil depletion in mice. In isolated murine neutrophils, long-chain polyphosphates modulated cytokine release induced by lipopolysaccharides (LPS) from Gram-negative bacteria or lipoteichoic acid from Gram-positive bacteria. In addition, long-chain polyphosphates induced immune evasive effects in human neutrophils. In detail, long-chain polyphosphates downregulated CD11b and curtailed the phagocytosis of Escherichia coli particles by neutrophils. Polyphosphates modulated the migration capacity by inducing CD62L shedding resulting in CD62Llow and CD11blow neutrophils. The release of IL-8 induced by LPS was also significantly reduced. Pharmacologic blockade of PI3K with wortmannin antagonized long-chain polyphosphate-induced effects on LPS-induced IL-8 release. In conclusion, polyphosphates govern immunomodulation in murine and human neutrophils, suggesting polyphosphates as a therapeutic target for bacterial infections to restore innate immune defense.

Modulation of Neutrophil Activity by Soluble Complement Cleavage Products—An In-Depth Analysis

The cellular and fluid phase-innate immune responses of many diseases predominantly involve activated neutrophil granulocytes and complement factors. However, a comparative systematic analysis of the early impact of key soluble complement cleavage products, including anaphylatoxins, on neutrophil granulocyte function is lacking. Neutrophil activity was monitored by flow cytometry regarding cellular (electro-)physiology, cellular activity, and changes in the surface expression of activation markers. The study revealed no major effects induced by C3a or C4a on neutrophil functions. By contrast, exposure to C5a or C5a des-Arg stimulated neutrophil activity as reflected in changes in membrane potential, intracellular pH, glucose uptake, and cellular size. Similarly, C5a and C5a des-Arg but no other monitored complement cleavage product enhanced phagocytosis and reactive oxygen species generation. C5a and C5a des-Arg also altered the neutrophil surface expression of several complement receptors and neutrophil activation markers, including C5aR1, CD62L, CD10, and CD11b, among others. In addition, a detailed characterization of the C5a-induced effects was performed with a time resolution of seconds. The multiparametric response of neutrophils was further analyzed by a principal component analysis, revealing CD11b, CD10, and CD16 to be key surrogates of the C5a-induced effects. Overall, we provide a comprehensive insight into the very early interactions of neutrophil granulocytes with activated complement split products and the resulting neutrophil activity. The results provide a basis for a better and, importantly, time-resolved and multiparametric understanding of neutrophil-related (patho-)physiologies.

Evaluating the Phagocytic Index of Peripheral Leukocytes in Endometriosis by Plasma Experiments.

Background and Objectives: Endometriosis is a benign, chronic disease, that negatively influences the quality of life of affected women and is responsible for a remarkable amount of infertility. The pathophysiology of the disease is still not clarified, but the insufficient immune surveillance plays a significant role in it. The phagocyte function of innate immune cells may play a role in the elimination of ectopic endometrium. The purpose of this study is to examine the phagocyte function of neutrophil granulocytes and monocytes, incubated in heat-inactivated and not-inactivated plasma samples from healthy women and from women with endometriosis before and after the surgical treatment. Materials and Methods: Blood samples were collected from eight preoperative and eight postoperative patients with endometriosis before and after the surgical treatment, and from 16 healthy patients as controls. Neutrophil granulocytes, monocytes and blood plasma samples were isolated. Cells were incubated in different plasma samples, and the phagocytic index was determined with a fluorescence microscope. Results: The phagocytic index of granulocytes and monocytes isolated from patients with endometriosis was significantly decreased compared to healthy women after the cells were incubated in their own plasma. Preoperatively isolated cells from patients with endometriosis demonstrated an improved phagocyte function after incubating them in plasma samples from healthy controls. In contrast, the phagocytic activity of cells from healthy women significantly reduced after being incubated in the plasma of preoperative endometriosis patients. The heat-inactivation of plasma samples did not affect the results. Conclusions: Active endometriosis lesions may produce heat-stable systemic immunomodulatory factors, which reduced the phagocyte function of peripheral monocytes and neutrophil granulocytes. The phagocyte function of these cells can be normalized after the complete surgical removal of endometriosis, which then demonstrates similar values as in healthy women.

HAX1-dependent control of mitochondrial proteostasis governs neutrophil granulocyte differentiation.

The relevance of molecular mechanisms governing mitochondrial proteostasis to the differentiation and function of hematopoietic and immune cells is largely elusive. Through dissection of the network of proteins related to HCLS1-associated protein X-1, we defined a potentially novel functional CLPB/HAX1/(PRKD2)/HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and, thus, elucidated molecular and metabolic mechanisms underlying congenital neutropenia in patients with HAX1 deficiency as well as bi- and monoallelic mutations in CLPB. As shown by stable isotope labeling by amino acids in cell culture (SILAC) proteomics, CLPB and HAX1 control the balance of mitochondrial protein synthesis and persistence crucial for proper mitochondrial function. Impaired mitochondrial protein dynamics are associated with decreased abundance of the serine-threonine kinase PRKD2 and HSP27 phosphorylated on serines 78 and 82. Cellular defects in HAX1–/– cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical molecular and metabolic mechanism governing the differentiation and function of neutrophil granulocytes.

Эффекты влияния рекомбинантного интерферона α-2b на фенотип субпопуляций нейтрофильных гранулоцитов пациентов с COVID-19

Investigation of molecular mechanisms associated with interferon (IFN) production and receptor function of neutrophil granulocytes (NGs) in COVID-19 is highly relevant because it can be promising in the search for new therapeutic strategies targeting NGs and their reactivity to restore and strengthen the innate immune response against SARS-CoV-2. Objective. To assess the effects of recombinant IFN-α2b on the phenotype of CD16+IFNα/βR1–CD119+, CD16+IFNα/βR1+CD119–, and CD16+IFNα/βR1+CD119+ NGs from peripheral blood of patients with COVID-19 in an in vitro experiment. Patients and methods. We analyzed blood samples from 31 patients with a mean age of 61 years (range: 57;71 years) with moderate COVID-19. We assessed the number of CD16+IFNα/βR1–CD119+, CD16+IFNα/βR1+CD119–, and CD16+IFNα/βR1+CD119+ NGs, receptor density (FC 500, ‘Beckman Coulter,’ USA), phagocytic activity of NGs before and after incubation with recombinant IFN-α2b. We also measured serum levels of several cytokines, including IFNα, IFNγ, IL-6, IL-8 (ELISA, ‘Vektor-Best’ LLC). The control group comprised 22 adult healthy individuals with a mean age of 58 years (range: 57; 70 years). Results. Patients with moderate COVID-19 demonstrated low serum levels of IFNα and IFNγ along with elevated levels of IL-6 and IL-8. We observed transformation of 3 phenotypes among NG subpopulations: CD16+IFNα/βR1–CD119+, CD16+IFNα/βR1+CD119-, and CD16+IFNα/βR1+CD119+. We observed positive remodulating effects of recombinant IFN-α2b on the number and phenotype of NG subpopulations and their phagocytic activity in our in vitro experiment. Conclusion. Recombinant IFN-α2b demonstrated positive effects in in vitro experiments; therefore, it can be considered in the future as a potential therapeutic tool for moderate COVID-19. Restoration of type I IFN might be an effective treatment option for COVID-19, because it can promote faster virus elimination, restore normal functioning of the IFN system, and have positive regulatory effects on the phenotype of NG subpopulations. Key words: neutrophil granulocytes, subpopulations, COVID-19, phenotype, recombinant interferon α2b

Induced Pluripotent Stem Cell-Derived Neutrophil Granulocytes - Flow Cytometry Analysis Reveals Distinct Subpopulations

Severe congenital neutropenia (SCN) comprises a spectrum of monogenic disorders characterized by impaired differentiation and function of neutrophil granulocytes. Since animal models often do not fully recapitulate human SCN phenotypes and primary bone marrow samples of patients are scarce, alternative strategies are desirable to study the genetic causes and mechanisms. Induced pluripotent stem cells (iPSCs) can be differentiated into neutrophil granulocytes, thereby presenting an excellent tool to study hematopoiesis and especially neutrophil differentiation in health and disease in vitro.Recently, neutrophil progenitors and mature neutrophils of murine and human bone marrow have been characterized by single-cell RNA sequencing, mass cytometry and flow cytometry and are now referred to as proNeu, preNeu, immature-Neu and mature-Neu, at least in part reflecting conventional morphological classification of myeloblasts, promyelocytes, myelocytes/metamyelocytes and band and segmented neutrophils, respectively.Here we developed a flow cytometry antibody panel and gating strategy, which robustly identified distinct myeloid subsets in iPS-derived neutrophils. We adopted a differentiation protocol, which consists of feeder- and serum-free differentiation of iPS cells by mesoderm induction and patterning, followed by lineage progression through hemogenic endothelium to hematopoietic progenitors and finally mature neutrophil granulocytes. Floating cells arise, which can be harvested continuously and analyzed by flow cytometry.Based on expression of cell surface molecules, we defined four subpopulations: After selecting for single, live, CD45 + and CD14 - cells, the different progenitor stages were first defined by their expression of CD117 and CD49d. CD117 midCD49d high cells were further stratified into SSC lowCD34 + cells and SSC highCD34 - cells, representing myeloblasts (proNeu1) and promyelocytes (proNeu2/preNeu), respectively. These cells progressed to CD117 -CD49d mid and were CD11b +CD101 +, which defines myelocytes/metamyelocytes (immature-Neus). CD117 -CD49d low cells were CD11b +CD101 + and expressed additionally CD16, resembling band/segmented neutrophils (mature-Neus). Additionally, these iPS-derived cells progressively expressed CD35, which is also a maturation marker of human myeloid cells in vivo. May-Grünwald-Giemsa staining of these four subpopulations (CD117 midCD49d highSSC lowCD34 +, CD117 mid CD49d highSSC highCD34 -, CD117 - CD49d midand CD117 - CD49d low) revealed homogenous populations of sorted cells, morphologically resembling myeloblasts, promyelocytes, myelocytes/metamyelocytes and band/segmented neutrophils, respectively.Ongoing studies in our lab make use of this model to a) validate the functional significance of rare genetic variants and b) further assess transcriptomic and proteomic changes on a single cell level. Thus, we provide a promising tool to study neutrophil differentiation in health and disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Kynurenic Acid and Its Analogue SZR-72 Ameliorate the Severity of Experimental Acute Necrotizing Pancreatitis

The pathophysiology of acute pancreatitis (AP) is not well understood, and the disease does not have specific therapy. Tryptophan metabolite L-kynurenic acid (KYNA) and its synthetic analogue SZR-72 are antagonists of the N-methyl-D-aspartate receptor (NMDAR) and have immune modulatory roles in several inflammatory diseases. Our aims were to investigate the effects of KYNA and SZR-72 on experimental AP and to reveal their possible mode of action. AP was induced by intraperitoneal (i.p.) injection of L-ornithine-HCl (LO) in SPRD rats. Animals were pretreated with 75-300 mg/kg KYNA or SZR-72. Control animals were injected with physiological saline instead of LO, KYNA and/or SZR-72. Laboratory and histological parameters, as well as pancreatic and systemic circulation were measured to evaluate AP severity. Pancreatic heat shock protein-72 and IL-1β were measured by western blot and ELISA, respectively. Pancreatic expression of NMDAR1 was investigated by RT-PCR and immunohistochemistry. Viability of isolated pancreatic acinar cells in response to LO, KYNA, SZR-72 and/or NMDA administration was assessed by propidium-iodide assay. The effects of LO and/or SZR-72 on neutrophil granulocyte function was also studied. Almost all investigated laboratory and histological parameters of AP were significantly reduced by administration of 300 mg/kg KYNA or SZR-72, whereas the 150 mg/kg or 75 mg/kg doses were less or not effective, respectively. The decreased pancreatic microcirculation was also improved in the AP groups treated with 300 mg/kg KYNA or SZR-72. Interestingly, pancreatic heat shock protein-72 expression was significantly increased by administration of SZR-72, KYNA and/or LO. mRNA and protein expression of NMDAR1 was detected in pancreatic tissue. LO treatment caused acinar cell toxicity which was reversed by 250 µM KYNA or SZR-72. Treatment of acini with NMDA (25, 250, 2000 µM) did not influence the effects of KYNA or SZR-72. Moreover, SZR-72 reduced LO-induced H2O2 production of neutrophil granulocytes. KYNA and SZR-72 have dose-dependent protective effects on LO-induced AP or acinar toxicity which seem to be independent of pancreatic NMDA receptors. Furthermore, SZR-72 treatment suppressed AP-induced activation of neutrophil granulocytes. This study suggests that administration of KYNA and its derivative could be beneficial in AP.

Dynamic Function and Composition Shift in Circulating Innate Immune Cells in Hibernating Garden Dormice.

Hibernation is characterized by successive torpor bouts during which metabolic rate is down-regulated to 2–4% of euthermic levels along with core body temperatures (Tb) ranging between 0 and 10°C. One characteristic of the torpid state, which is periodically interrupted by a few hours of euthermic phases or arousals during hibernation, resides in an overall impairment of the immune system. The most striking change during torpor is the reduction of circulating white blood cells up to 90%, while their numbers rise to near summer euthermic level upon rewarming. However, potential changes in responsiveness and function of neutrophil granulocytes, accounting for the primary cellular innate immune defense, are unknown. Here we present the first data on shifts in oxidative burst capacity, i.e., the ability to produce reactive oxygen species (ROS), of neutrophils during hibernation. Using a chemiluminescence assay, we measured real-time ROS production in whole blood of hibernating garden dormice (Eliomys quercinus) in early or late torpor, and upon arousals. Accounting for changes in neutrophil numbers along the torpor-arousal cycle, we found significant differences, between torpid and euthermic states, in the neutrophil oxidative burst capacity (NOC), with shallow cell responses during torpor and a highly significant increase by up to 30-fold during arousals. Further, we observed a significant reduction of NOC from aroused animals with euthermic Tb of 36.95 ± 0.37°C, when tested at 6°C, whereas no change occurred in NOC from torpid individuals reaching constant Tb of 4.67 ± 0.42°C, when measured at 35°C. This dynamic indicates that the reduction in NOC during torpor may be temperature-compensated. These results linked to the understanding of immune function during the torpor-arousal cycle might have clinical relevance in the context of therapeutic hypothermia and reperfusion injury.

Phagocyte function of peripheral neutrophil granulocytes and monocytes in endometriosis before and after surgery

IntroductionEndometriosis is a chronic systemic disease, which influence negatively the quality of life of affected women and responsible for infertility and chronic pelvic pain. Pathophysiology of the disease is still enigmatic, but insufficient immune surveillance may play a role in it. Peripheral natural immune cell function is rarely examined. The aim of the study was to examine phagocyte function of peripheral neutrophil granulocytes and monocytes, whether this phagocytic activity is affected by the presence or removal of endometriotic lesions in women with endometriosis. Material and methodsTwenty-six preoperative, 13 postoperative samples from women with endometriosis, 23 samples from healthy women, 14 pre- and postoperative samples from the surgical control group were enrolled. Cells were isolated from peripheral blood samples, marked and evaluated for the phagocytosis index with immunofluorescent microscope after phagocyting the zymosane molecules. ResultsPhagocyte function of monocytes and neutrophil granulocytes decreased significantly women with endometriosis before surgery compared to healthy controls. However, 7 days after surgery the postoperative values showed significant improvement compared to the preoperative results of women with endometriosis. This increment reached the values of the healthy women. In the surgical control group no difference was detected between the pre- and postoperative outcomes. DiscussionDecreased phagocyte function of the examined cells, which can be the result of the circulating immunosuppressive factors, may play a role in the deficient clearance of ectopic endometrial tissue. Based on the postoperative results, these immunosuppressive factors may be reduced or eliminated 7 days after surgery in women with endometriosis.

Early Post-Stroke Infections Are Associated with an Impaired Function of Neutrophil Granulocytes.

To investigate whether neutrophil granulocytes’ function relates to post-stroke infections and clinical outcome after stroke, we prospectively recruited 95 patients after ischemic stroke and tested them for their microbiocidal neutrophil functions in this exploratory study. Additionally, 24 age-adjusted controls were examined regarding neutrophil function. Phagocytic capacity and the ability of the neutrophil granulocytes to produce reactive oxygen species (ROS) as well as CD11b and CD16 receptor expression profile were measured by flow cytometry at days 1, 3, 7, and 90 after symptom onset. Primary outcome was the development of an infection within the first week after stroke. Results of neutrophil functional measurements were compared between patients with and without infection as well as between all stroke patients and controls. Further risk factors for the development of infections were summarized in an infection-risk score for the purpose of multivariate statistical analysis. The ROS production in neutrophils after stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) was reduced at baseline in patients with post-stroke infections compared to those without (p = 0.013). This difference proved to be independent from the infection-risk score in the binary logistic regression (p = 0.011). Phagocytosis and oxidative bursts were not significantly reduced in the whole stroke patient group compared to controls. Dysfunction of neutrophil granulocytes seems to play a significant role in the development of post-stroke infections. Further studies are warranted to investigate neutrophil granulocytes´ function as a potential biomarker of post-stroke infections.

Патогенетическое обоснование применения иммунотерапии в лечении рекуррентных ко-инфекций верхних дыхательных путей у детей с врожденными расщелинами губы и нёба

The synergism of the action of co-infecting microorganisms contributes to their overcoming the epithelial barrier, modification of the function of cells of the immune system (IS) and evading the immune response. In children with congenital cleft lip and palate (CCLP), despite elimination of the anatomical defect at an earlier age, the frequency of infectious and inflammatory diseases of the respiratory tract and oral cavity involving pathogen associations increases as they get older, at the same time defects in the functioning of IS are preserved. Objective. To develop immunotherapy program for children with CCLP, suffering from recurrent co-infections of the upper respiratory tract and oral cavity, based on the study of the dynamics of the microbial landscape transformation, IS dysfunction, clinical manifestations of infectious and inflammatory diseases of the upper respiratory tract and oral cavity in children with CCLP at different ages. Patients and methods. The study included 120 children from 1 to 12 years of age with CCLP at different stages of surgical treatment. The study was carried out: T and B lymphocytes, natural killer cells (FC500 "Beckman Coulter", USA), phagocytic and microbicidal functions of neutrophilic granulocytes (NG), serum IgA, IgM, IgG and sIgA levels in the oral fluid (ELISA), detection of microbial pathogens (microbiological analysis, ELISA) and viral (PCR). Results. Comprehensive examination of children with CCLP at different age periods, including characteristics of the clinical manifestations of infectious and inflammatory diseases, assessment of the microbial landscape of the oral cavity and upper respiratory tract with the identification of co-infection features, clarification of the variants of IS dysfunction, made it possible to reveal children with the most severe clinical manifestations of recurrent co-infections and to optimize treatment. Conclusion. The developed program of combined local interferon and systemic immunotherapy for immunocompromised children with CCLP contributes to the restoration of IS functions, anti-infective resistance and achievement of positive clinical effects in the form of regression of clinical signs of immunocompromise in patients with recurrent co-infections, which allows to obtain a protective effect and provide timely surgical care, to avoid complications in the postoperative period and at the stage of recovery, and by improving anti-infective immunity to enhance the quality of life of children with CCLP. Key words: children, congenital cleft lip and palate, co-infection, systemic immunity, microbiocenosis, immunotherapy

Defects in Signal Recognition Particle (SRP) Components Reveal an Essential and Non-Redundant Role for Granule Biogenesis and Differentiation of Neutrophil Granulocytes

Neutrophil granulocyte play pivotal roles in inflammatory responses, immune defence, tissue remodeling, and cancer control. Studying rare patients with defects in differentiation and/or function of neutrophil granulocytes highlights genes and pathways orchestrating these important cellular functions. A previously not appreciated role of the signal recognition particle (SRP) has emerged when monoallelic mutations in SRP54 were associated with congenital neutropenia and pancreatic insufficiency. The eukaryotic SRP is composed of six distinct polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68, SRP72) bound to an RNA molecule (the 7SL RNA). SRP and its receptor (SRPRA and SRPRB) cooperatively transport nascent proteins emerging from ribosome to the endoplasmic reticulum (ER) lumen via a protein-conducting channel. We hypothesized that other constituents of the SRP may be involved in the pathophysiology of congenital neutropenia and that disturbances of the SRP may result in defective proteostasis in neutrophil granulocytes. By virtue of our international Care-for-Rare Alliance we identified three kindred with molecularly unsolved congenital neutropenia. Exome-sequencing revealed a de novo monoallelic mutation in SRPRA in one individual in one family and a homozygous splice-site mutation in SRP19 in five individuals from two families. Clinically, all patients with mutations in SRPRA and SRP19 suffered from various degrees of neutropenia and susceptibility to bacterial infections. Pancreatic insufficiency was only seen in patients with mutation in SRPRA, whereas SRP19 deficiency was not associated with any non-hematopoietic symptoms. To functionally validate these novel genetic variants, we set up an in-vitro neutrophil differentiation system allowing us to model the genetic defects in human SRP complex subunits. We introduced patient-specific mutations (SRPRA Q464E and SRP19 c.189+5G&amp;gt;A) into wildtype iPS cells using CRISPR-Cas9 editing. The engineered cells were differentiated into bona fide neutrophil granulocytes. In vitro generated wildtype neutrophil granulocytes had a high degree of resemblance to primary peripheral blood neutrophil granulocytes, as evidenced by phenotypic (light microscopy, cell surface markers, gene expression) and functional (phagocytosis, bacterial killing, rolling and adhesion, chemotaxis) characteristics. In striking contrast to wildtype iPS cells, both SRPRA and SRP19 mutated iPS cells had a significantly reduced capacity to differentiate into neutrophil granulocytes, as shown by colony-forming unit assays and phenotypic analysis. Furthermore, in vitro generated neutrophil granulocytes had higher susceptibility to apoptosis and evidence of increased unfolded protein responses. We went on to characterize the proteome of primary neutrophil granulocytes from patients with SRP-defects by data-independent proteomics technology. Interestingly, neutrophil granulocytes with defects in SRP constituents shared a significantly reduced abundance of the proteins Pentraxin-3 (PTX3) and Glucuronidase Beta (GUSB). These findings were confirmed in both non-hematopoietic HELA cells and promyelocytic HL60 leukemia cells engineered to express the respective SRP mutants. Upon genetic reconstitution, the aberrant protein expression was re-established. In summary, our studies define novel genetic defects causing congenital neutropenia and highlight the relevance of coordinated protein trafficking pathways and homeostasis for physiological differentiation and function of human neutrophil granulocytes. Disclosures No relevant conflicts of interest to declare.

Proteome Analysis of Human Neutrophil Granulocytes From Patients With Monogenic Disease Using Data-independent Acquisition

Neutrophil granulocytes are critical mediators of innate immunity and tissue regeneration. Rare diseases of neutrophil granulocytes may affect their differentiation and/or functions. However, there are very few validated diagnostic tests assessing the functions of neutrophil granulocytes in these diseases. Here, we set out to probe omics analysis as a novel diagnostic platform for patients with defective differentiation and function of neutrophil granulocytes. We analyzed highly purified neutrophil granulocytes from 68 healthy individuals and 16 patients with rare monogenic diseases. Cells were isolated from fresh venous blood (purity >99%) and used to create a spectral library covering almost 8000 proteins using strong cation exchange fractionation. Patient neutrophil samples were then analyzed by data-independent acquisition proteomics, quantifying 4154 proteins in each sample. Neutrophils with mutations in the neutrophil elastase gene ELANE showed large proteome changes that suggest these mutations may affect maturation of neutrophil granulocytes and initiate misfolded protein response and cellular stress mechanisms. In contrast, only few proteins changed in patients with leukocyte adhesion deficiency (LAD) and chronic granulomatous disease (CGD). Strikingly, neutrophil transcriptome analysis showed no correlation with its proteome. In case of two patients with undetermined genetic causes, proteome analysis guided the targeted genetic diagnostics and uncovered the underlying genomic mutations. Data-independent acquisition proteomics may help to define novel pathomechanisms in neutrophil diseases and provide a clinically useful diagnostic dimension.

Children with rare diseases of neutrophil granulocytes: from therapeutic orphans to pioneers of individualized medicine

Neutrophil granulocytes are the most abundant immune cells in the blood yet the pathways orchestrating their differentiation and biological function remain incompletely understood. Studying (ultra-) rare patients with monogenetic defects of neutrophil granulocytes may open new horizons to understand basic principles of hematopoiesis and innate immunity. Here, recent insights into genetic factors controlling myelopoiesis and their more general role in biology will be presented in a clinical perspective. Advances in supportive care, first and foremost the use of recombinant human granulocyte-colony stimulating factor, has made a substantial difference for the quality of life and life expectancy of patients with congenital neutropenia (CN). Up to date, the only definitive cure can be provided by transplantation of allogeneic hematopoietic stem cells. The elucidation of the underlying molecular factors contributing to defective differentiation and function of neutrophil granulocytes nurtures new ideas of targeted individualized therapies.

The effect of healthy pregnant plasma and preeclamptic plasma on the phagocytosis index of neutrophil granulocytes and monocytes of nonpregnant women

ABSTRACTObjective: Phagocyte function of neutrophil granulocytes and monocytes is decreased in healthy pregnancy and further decreased in preeclampsia. The cause of the declined function is unknown. Decreased phagocyte function can lead to the higher infection rate in healthy pregnancy and may also be responsible for the increased susceptibility to infections and high trophoblast concentration in preeclampsia. The aim of this study is to examine the phagocyte function of neutrophil granulocytes and monocytes. Methods: Monocytes and neutrophil granulocytes were separated from the peripheral circulation of six nonpregnant patients and incubated in plasma samples from six healthy pregnant, six preeclamptic pregnant, and six nonpregnant patients. The cells were marked and evaluated for the phagocytosis index with immunofluorescent microscope after phagocyting the zymosan molecules. Results: Phagocyte function of neutrophils as well as monocytes from nonpregnant patients were decreased significantly when the cells were incubated in plasma samples from healthy pregnant patients, and further decreased when incubated in plasma samples from preeclamptic pregnant women. Conclusion: The decreased phagocyte function of neutrophil granulocytes and that of monocytes in healthy pregnancy and the further decreased phagocyte function in preeclampsia is caused by factor(s) in the maternal circulation.

CHARACTERISTICS NONSPECIFIC ELEMENT IMMUNITY IN PATIENTS WITH CHRONIC ATROPHIC GASTRITIS

Background: Investigate characteristics non-specific immunity in the blood in patients with chronic gastritis and chronic atrophic gastric. Materials and methods: The study involved 200 patients aged 25 to 65 who were in randomly into 3 groups. The first group included 100 healthy subjects in the second group – 85 patients with moderate atrophic gastritis, gastric body, the third group – 25 patients with severe atrophic gastritis of the body of the stomach. The study was conducted with the approval of the Ethics Committee FGBNU “Institute of Medical Problems of the North” (Acting Director – MD, professor Kasparov E.V.). Each participant signed an informed consent form for examination, according to the Declaration of Helsinki of the World Medical Association, which regulates conduct research. Diagnosis of atrophic gastritis performed by determining the pepsinogen-1, 2-pepsinogen and H. pylori antibodies in serum and morphological study of the gastric mucosa. Evaluation of non-specific immunity was conducted by studying the chemiluminescent activity of neutrophils (De sole p.et al.; 1983). Statistical analysis was performed using the application package Statistica 7.0 (StatSoft, USA). Results: violation of spontaneous and induced functional activity of neutrophils observed in all groups of patients with chronic gastritis, while in patients with chronic superficial gastritis revealed a reduction in both spontaneous and induced chemiluminescence activity of neutrophils. On the other side of the milestones of patients with chronic atrophic gastritis there is an increase of the functional activity of neutrophils expressed significantly more severe atrophic changes in the gastric mucosa. Considering that the main function of neutrophil granulocytes – phagocytosis of bacterial agents, modified own cells, their increased activity can be attributed to an infectious process in the gastric mucosa and harsh destruction of cells of the stomach due to atrophic changes. Conclusion: thus, with an increase in infectious load or atrophic changes in the mucosa of the stomach does not exclude the exhaustion of reserve capacity of phagocytes, their modification, thus the transformation of precancerous condition in cancer.