Polymorphonuclear Cell Function Research Articles

Polymorphonuclear myeloid-derived suppressor cells play a proinflammatory role via TNF-α+ B cells through BAFF/BTK/NF-κB signalling pathway in the pathogenesis of collagen-induced arthritis mice.

Although various studies have been performed on the function of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in RA, the results were conflicting. Here we were trying to clarify the role of PMN-MDSCs in the pathogenesis of RA and its specific mechanisms. We detected the frequencies and counts of PMN-MDSCs, TNF-α+ B cells and Ki67+ B cells in spleen and inflamed joints of collagen-induced arthritis (CIA) mice using flow cytometry. The pathological role of PMN-MDSCs was examined by anti-Ly6G neutralizing antibodies against PMN-MDSCs or adoptive transfer of PMN-MDSCs. And the modulation of PMN-MDSCs on B cells was conducted by coculture assays, RNA-Seq, RT-qPCR, and so on. The mechanism of BAFF regulating B cells was verified through western blot and flow cytometry. PMN-MDSCs accumulated in the spleen and joints of CIA mice. PMN-MDSCs depletion could alleviate the arthritis severity, which was accompanied by decreased TNF-α secretion and proliferation of B cells. And its adoptive transfer also facilitated disease progress. Furthermore, PMN-MDSCs from CIA mice had higher expression level of BAFF, which regulated TNF-α expression, proliferation and apoptosis of B cells in vitro. What's more, BAFF promoted phosphorylation of BTK/NF-κB signalling pathway. And Ibrutinib (BTK inhibitor) could reverse the effect of BAFF on TNF-α expression of B cells. Our study suggested that PMN-MDSCs enhanced disease severity of CIA and manipulated TNF-α expression, proliferation and apoptosis of B cells via BAFF, furthermore, BAFF promoted TNF-α expression through BTK/NF-κB signalling pathway, which demonstrated a novel pathogenesis of PMN-MDSCs in CIA.

CD47-SIRPα Controls ADCC Killing of Primary T Cells by PMN Through a Combination of Trogocytosis and NADPH Oxidase Activation

Immunotherapies targeting the “don’t eat me” myeloid checkpoint constituted by CD47 SIRPα interaction have promising clinical potential but are limited by toxicities associated with the destruction of non-tumor cells. These dose-limiting toxicities demonstrate the need to highlight the mechanisms of anti–CD47-SIRPα therapy effects on non-tumor CD47-bearing cells. Given the increased incidence of lymphopenia in patients receiving anti-CD47 antibodies and the strong ADCC (antibody-dependent cellular cytotoxicity) effector function of polymorphonuclear cells (PMNs), we investigated the behavior of primary PMNs cocultured with primary T cells in the presence of anti-CD47 mAbs. PMNs killed T cells in a CD47-mAb–dependent manner and at a remarkably potent PMN to T cell ratio of 1:1. The observed cytotoxicity was produced by a novel combination of both trogocytosis and a strong respiratory burst induced by classical ADCC and CD47-SIRPα checkpoint blockade. The complex effect of the CD47 blocking mAb could be recapitulated by combining its individual mechanistic elements: ADCC, SIRPα blockade, and ROS induction. Although previous studies had concluded that disruption of SIRPα signaling in PMNs was limited to trogocytosis-specific cytotoxicity, our results suggest that SIRPα also tightly controls activation of NADPH oxidase, a function demonstrated during differentiation of immature PMNs but not so far in mature PMNs. Together, our results highlight the need to integrate PMNs in the development of molecules targeting the CD47-SIRPα immune checkpoint and to design agents able to enhance myeloid cell function while limiting adverse effects on healthy cells able to participate in the anti-tumor immune response.

Evaluation of peripheral blood polymorphonuclear cell functions after an oral carbohydrate overload in obese and insulin dysregulated horses

Obesity and insulin dysregulation (ID) are increasingly prevalent conditions in equid populations worldwide. Immune impairment is well described in humans with metabolic dysfunction and is reported but still incompletely understood in horses. This study evaluated the effect of acute induced transient hyperglycemia on apoptosis, phagocytosis and oxidative burst activity of peripheral blood polymorphonuclear cells (PMN) of lean and obese adult horses with or without insulin dysregulation. Seventeen adult horses were allocated into three groups based on their body condition score (BCS) and metabolic status: lean-insulin sensitive (lean-IS), obese-insulin sensitive (obese-IS) and obese-insulin dysregulated (obese-ID). ID was determined by insulin tolerance testing (ITT). Blood glucose elevation was induced through an infeed-oral glucose test (in-feed OGT), and all assessments of PMN functions (apoptosis, phagocytosis and oxidative burst) were done in vitro after isolation from peripheral blood before and 120 min after carbohydrate overload. Results were analyzed using a repeated measures linear mixed model with significance defined at P < 0.05. No differences in apoptosis were observed between experimental groups at any time point. Phagocytic capacity was significantly lower at baseline in the obese-ID group but increased in response to glucose administration when compared to the other two groups. Basal reactive oxygen species production in the obese-IS group differed significantly from the lean-IS and obese-ID groups and decreased significantly in response to glucose administration. Results from this study showed that both metabolic status itself, and oral glucose administration, seem to be factors that alter PMN functionality in horses, specifically phagocytosis and oxidative burst.

Alcohol influences gene expression and function of polymorphonuclear cells in liver disease

Background and Aim: Alcohol is one of the major causes of liver cirrhosis worldwide. Patients with alcohol-related liver diseases are highly predisposed to infection. It is reported that, neutrophils counts are higher in peripheral blood and liver tissue of these patients. Neutrophils are the first responders to infection and tissue injury and optimal neutrophil function is important in disease resolution. Thus, our objectives were to investigate the effects of alcohol on neutrophil function, gene expression and ability to resolve infection.

PSI-1 Effects of choline on immune cell function in growing cattle supplemented with guanidinoacetic acid and creatine

Abstract Methyl sources that affect immune function/inflammation potentially have value in the beef industry, notably with receiving cattle. In newly received beef cattle, an active immune response with appropriately controlled inflammation is critical to optimize both biological and economic efficiency, due to the high cost of disease and its treatment. Our objective was to determine how modulation of methyl group status would affect polymorphonuclear cell (PMN; primarily neutrophils) function. Six ruminally cannulated Holstein steers (200 kg) were used in a 6×6 Latin square design with 10-d periods. Factorial treatments included 3 methyl group modulators (MGM: control; 15 g/d guanidinoacetic acid, which consumes methyl groups; or 16.8 g/d creatine, which spares methyl groups) and 2 levels of choline (0 or 5 g/d). Steers received 4 kg/d of a corn-based diet. Treatments were continuously infused abomasally. On d 10 of each period, jugular blood was collected, and PMN were isolated. Cells were challenged with or without LPS (1 μg/mL) for 30 min followed by addition of dihydrorhodamine (100 μM) for 10 min and E. coli covalently labeled with Texas Red at a ratio of 20:1 for an additional 40 min. PMN activity (phagocytosis and oxidative burst) was assessed using a capillary flow cytometer. Oxidative burst, with or without LPS challenge, was not affected by choline (P &amp;gt;0.42) or MGM (P &amp;gt;0.75). Phagocytosis without LPS was not affected by choline (P=0.29) or MGM (P=0.30). When PMN were challenged with LPS, phagocytosis tended to be reduced by choline (P=0.09) but was not affected by MGM (P=0.35). Choline tended to reduce phagocytosis by PMN challenged with LPS, suggesting that it may reduce inflammatory responses. A more robust understanding of methyl group metabolism may allow nutritionists to supply these nutrients to optimize immune function.

Polymorphonuclear Cell Chemotaxis and Suicidal NETosis: Simultaneous Observation Using fMLP, PMA, H7, and Live Cell Imaging.

Chemotaxis and the formation of suicidal neutrophil extracellular traps (suicidal NETosis) are key functions of polymorphonuclear cells (PMNs). Neutrophil extracellular traps in particular are known to be significantly involved in the severity of inflammatory and immunological disorders such as rheumatoid arthritis and Crohn's disease. Therefore, detailed knowledge of PMNs is essential for analyzing the mechanisms involved in, and developing new therapies for, such diseases. To date, no standard method to analyze these cell activities has been established. This study used in vitro live cell imaging to simultaneously observe and analyze PMN functions. To demonstrate this, the effects of phorbol-12-myristat-13-acetat (PMA, 0.1-10 nM), N-formylmethionine-leucyl-phenylalanine (fMLP, 10 nM), and protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) on PMN chemotaxis and suicidal NETosis were studied. PMA (1 nM-10 nM) resulted in significant concentration-dependent behavior in chemotaxis and an earlier onset of maximum oxidative burst and NET formation of up to 44%. When adding H7, PMA-triggered PMN functions were reduced, demonstrating that all three functions rely mostly on protein kinase C (PKC) activity, while PKC is not essential for fMLP-induced PMN activity. Thus, the method here described can be used to objectively quantify PMN functions and, especially through the regulation of the PKC pathway, could be useful in further clinical studies of immunological disorders.

Suppression of Mll1-Complex by Stat3/Cebpβ-Induced miR-21a/21b/181b Maintains the Accumulation, Homeostasis, and Immunosuppressive Function of Polymorphonuclear Myeloid-Derived Suppressor Cells.

Mixed-lineage leukemia 1 (MLL1), which exerts its H3K4 methyltransferase activity by interacting with WDR5, ASH2L, and RBBP5, plays a pivotal role in regulating hematopoietic stem cell homeostasis. Disrupting the integrity of MLL1-complex has been reported to be associated with acute leukemia. However, the exact role of MLL1-complex in myeloid cells is unknown. In this study, microarray analysis revealed that the core components of the Mll1-complex, Wdr5, Ash2l, and Mll1, were concurrently downregulated by tumor-secreted factors as well as GM-CSF + IL-6 during the accumulation and activation of murine myeloid-derived suppressor cells (MDSCs). These changes were further validated by quantitative RT-PCR and Western blotting both in vitro and in vivo. The expression levels of WDR5 and ASH2L were also significantly decreased in bone marrow MDSCs of lung cancer patients compared with that of healthy controls. Functionally, ectopic expression of Wdr5, Ash2l, and Mll1 (C terminus) reversed the accumulation and function of GM-CSF + IL-6-induced as well as tumor-cocultured polymorphonuclear MDSCs (PMN-MDSCs) by promoting them to differentiate into mature neutrophil-like cells. Mechanistically, GM-CSF + IL-6-activated Stat3 and Cebpβ synergistically induced the expression of miR-21a, miR-21b, and miR-181b, and thus inhibited the expression of Wdr5, Ash2l, and Mll1 by targeting to their 3' untranslated regions, respectively. Furthermore, knockdown of these microRNAs also suppressed the expansion and function of GM-CSF + IL-6-induced PMN-MDSCs. Taken together, our findings indicate that the Stat3/Cebpβ-miR-21a/b/181b-Mll1-complex axis may play a critical role in PMN-MDSC expansion, activation, and differentiation, and this axis may provide an effectively immunological therapeutic approach for patients with cancer or other immunological diseases.

Oral vitamin C treatment increases polymorphonuclear cell functions in type 2 diabetes mellitus patients with poor glycemic control

This study investigated the effect of vitamin C on polymorphonuclear (PMN) cell functions in type 2 diabetes mellitus patients with poor glycemic control. We hypothesized that oral vitamin C treatment improves PMN cell functions. Patients (14) received either a vitamin C (1000 mg/d) or placebo (anhydrous calcium hydrogen phosphate) tablet for 6 weeks and were subjected to a 6-week washout period followed by a 6-week treatment crossover period. Blood samples were collected at pretreatment and posttreatment for PMN cell functions (by flow cytometry) and plasma vitamin C concentration. Phagocytosis was examined by incubating whole blood samples with fluorescein isothiocyanate–labeled Staphylococcus aureus, and oxidative burst was simultaneously evaluated by adding hydroethidine. In comparison with placebo, vitamin C increased both PMN cell phagocytosis (pretreatment: placebo, 17.8% ± 1.6% and vitamin C, 19.0% ± 3.4%, P = .70; posttreatment: placebo, 16.6% ± 1.7% and vitamin C, 27.1% ± 2.9%, P = .005) and oxidative burst (pretreatment: placebo, 6.4% ± 0.8% and vitamin C, 7.1% ± 1.2%, P = .60; posttreatment: placebo, 6.9% ± 1.3% and vitamin C, 12.1% ± 1.6%, P = .02). The plasma vitamin C concentration was elevated after vitamin C treatment as compared with that before treatment (P < .001) and was higher than that observed in the placebo treatment group (P < .01). Plasma vitamin C concentration and PMN cell functions were not significantly different before both treatments. We conclude that the 6-week 1000-mg/d vitamin C increased PMN phagocytosis and oxidative burst in type 2 diabetes mellitus patients with poor glycemic control.

LncRNA AK036396 inhibits maturation and accelerates immunosuppression of polymorphonuclear myeloid-derived suppressor cells by enhancing the stability of ficolin B

Abstract Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of cell biology. However, the role of lncRNAs in the development and function of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) remains unclear. Here, we identified that the lncRNA AK036396 was highly expressed in PMN-MDSCs, and lncRNA AK036396 knockdown promoted the maturation and decreased the suppressive function of PMN-MDSCs. Ficolin B (Fcnb), whose expression could be used to reflect PMN-MDSC development, was the predicted target gene of lncRNA AK036396 based on microarray results. LncRNA AK036396 knockdown attenuated Fcnb protein stability in a manner dependent on the ubiquitin-proteasome system. Moreover, Fcnb inhibition downregulated the suppressive function of PMN-MDSCs. In addition, the expression of human M-ficolin, which is an orthologue of mouse Fcnb, was increased and positively correlated with arginase1 (Arg1) level in lung cancer patients. Based on these findings, lncRNA AK036396 can inhibit maturation and accelerate immunosuppression of PMN-MDSCs by enhancing Fcnb protein stability.

LncRNA AK036396/FcnB regulating polymorphonuclear myeloid-derived suppressor cells in tumor bearing mice

Abstract Myeloid-derived suppressor cells (MDSCs), which are constituted of immature myeloid cells, represent a population of heterogeneous cells derived from bone marrow. Activated and expanded MDSCs suppress the antitumor immune response by directly inhibiting T cell by high levels of Arg1, reactive oxygen species, and inducible nitric oxide synthase. Long non-coding RNAs (lncRNAs) are transcripts with lengths &amp;gt;200 nts and without the capacity to encode proteins. With the development of RNA sequencing, lncRNAs that were previously considered “transcriptional noise” have been confirmed to be involved in regulating cellular biology. However, the role of lncRNAs in the development and function of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) remains unclear. Here, we identified that the lncRNA AK036396 was highly expressed in PMN-MDSCs, and lncRNA AK036396 knockdown promoted the maturation and decreased the suppressive function of PMN-MDSCs. Ficolin B (Fcnb), whose expression could be used to reflect PMN-MDSC development, was the predicted target gene of lncRNA AK036396 based on microarray results. LncRNA AK036396 knockdown attenuated Fcnb protein stability in a manner dependent on the ubiquitin-proteasome system. Moreover, Fcnb inhibition downregulated the suppressive function of PMN-MDSCs. In addition, the expression of human M-ficolin, which is an orthologue of mouse Fcnb, was increased and positively correlated with arginase1 (Arg1) level in lung cancer patients. Based on these findings, lncRNA AK036396 can inhibit maturation and accelerate immunosuppression of PMN-MDSCs by enhancing Fcnb protein stability, which will be helpful for developing antitumor therapies targeting PMN-MDSCs.

LncRNA AK036396 Inhibits Maturation and Accelerates Immunosuppression of Polymorphonuclear Myeloid-Derived Suppressor Cells by Enhancing the Stability of Ficolin B.

Long noncoding RNAs (lncRNA) are emerging as crucial regulators of cell biology. However, the role of lncRNAs in the development and function of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) remains unclear. Here, we identified that the lncRNA F730016J06Rik (AK036396) was highly expressed in PMN-MDSCs and that lncRNA AK036396 knockdown promoted the maturation and decreased the suppressive function of PMN-MDSCs. Ficolin B (Fcnb), the expression of which could be assessed as a surrogate for PMN-MDSC development, was the predicted target gene of lncRNA AK036396 based on microarray results. LncRNA AK036396 knockdown attenuated Fcnb protein stability in a manner dependent on the ubiquitin-proteasome system. Moreover, Fcnb inhibition downregulated the suppressive function of PMN-MDSCs. In addition, the expression of human M-ficolin, which is an ortholog of mouse Fcnb, was increased and positively correlated with arginase1 (ARG1) expression. This suppressive molecule is released by MDSCs, and its production is commonly used to represent the suppressive activity of MDSCs in patients with lung cancer, suggesting clinical relevance for these findings. These results indicate that lncRNA AK036396 can inhibit maturation and accelerate immunosuppression of PMN-MDSCs by enhancing Fcnb protein stability.

Assessment of heat stress induced alterations in polymorphonuclear (PMN) cell activity in native and crossbred cows

ABSTRACT Our current investigation was aimed to study differential influence of heat stress on defensive functions of blood polymorphonuclear cells among native and crossbred cows by culturing them in vitro after animal exposure to in vivo heat stress. Blood was collected from 18 cows which were divided in to three groups based on their breed as, group I (Sahiwal), group II (Tharparkar) and group III (Crossbred). THI was calculated by temperature and relative humidity (RH). PMN were isolated and were incubated at 38°C. Phagocytosis and lymphocyte proliferation was then investigated by NBT and MTT assays, respectively. The overall mean THI was significantly (p < 0.5) higher during summer (88.41 ± 1.54) when compared to the winter season (64.75 ± 0.97). There is a significant high lymphocytic proliferation in Group II when compared to Group III cattle. Also, there is a significant decrease in PA during the summer when compared to winter in all the breeds and in group III there is significant decrease in LPA during summer. The results of the present study suggest that indigenous cattle Tharparkar exhibited more tolerance to heat stress than the Group-I and Group-III during summer. However, in winter, all the breeds showed similar.

Technical note: Assessment of neutrophil endocytosis and proteolytic degradation and its relationship with phagocytosis and oxidative burst in dairy cows

Conventional assays of polymorphonuclear cell (PMN, neutrophil) function such as oxidative burst (OB) and phagocytosis (PC) are widely used to evaluate innate immunity in the transition period of dairy cows. Oxidative burst is commonly evaluated by measuring PMN median fluorescence intensity (MFI) involving the release of reactive oxygen species after in vitro stimulation. Phagocytosis can be measured by engulfment of fluorescent beads by PMN. DQ-ovalbumin (DQ-OVA) is a molecule suitable for the assessment of intracellular proteolytic degradation, so it might be informative about an additional pathway of pathogen handling by PMN. In this study, we evaluated the use of the DQ-OVA assay for the assessment of PMN function and the relationships among OB, PC, and DQ-OVA results in PMN isolated from blood of dairy cows between 5 and 21 d post partum. Results of the DQ-OVA validation assay were assessed with mixed linear regression models. Pearson correlation tests and kappa values for agreement were used to associate the MFI between each PMN function assay (OB, PC, and DQ-OVA). For the validation assay (9 cows in 3 replicates), PMN incubated with DQ-OVA were stimulated with IFN-γ or inhibited with cytochalasin D, and fluorescence was compared with untreated PMN. Stimulated and inhibited PMN had greater (970 ± 160 units) and lesser (593 ± 55 units) MFI relative to untreated PMN (791 ± 154 units), respectively, indicating that DQ-OVA fluorescence reflected enhanced or reduced endocytic and proteolytic function. To associate the MFI outcomes among OB, PC, and DQ-OVA, 153 samples from 40 cows were analyzed. Results showed significant, although weak association between DQ-OVA and PC MFI (Pearson r = 0.16). When values of MFI were categorized according to the first ("high" PMN functionality), second and third ("moderate" PMN functionality), or fourth ("low" PMN functionality) quartiles, agreement beyond chance (κ) was moderate: κ = 0.38 for DQ-OVA and OB, κ = 0.43 for DQ-OVA and PC, and κ = 0.43 for OB and PC. The DQ-OVA assay may complement traditional PMN functional assays because it provides additional information regarding the combination of endocytosis and proteolytic degradation, but it is not a substitute for assessment of OB or PC.

Proton-pump inhibitors elevate infection rate in cardiothoracic surgery patients by influencing PMN function in vitro and in vivo.

Proton-pump inhibitors (PPI) as pantoprazole are highly effective acid suppressive agents that belong to the world's most sold medication. However, they are pronounced to have immunosuppressive aspects. In our study, a negative influence of PPI on functions of polymorphonuclear cells in vitro like phagocytosis, oxidative burst, chemotaxis, and killing activity was shown, whereas formation of neutrophil extracellular traps (NET)osis remained unaffected. Pantoprazole stimulation additionally reduced the production of the proinflammatory cytokine IL-1β in whole blood assay as well as the production of IL-2 and IFN-γ after whole blood stimulation with phytohaemagglutinin. Moreover, IFN-γ feedback mechanisms and signaling by STAT-1 was impaired by PPI. Cardiac surgery is accompanied by developing systemic inflammatory response syndrome with immunosuppressive aspects. We exhibited reduced oxidative burst analyzing cardiac surgery patients' samples receiving or not receiving PPI. Furthermore, a higher rate of infections in patients receiving permanent PPI medication in retrospective analysis was uncovered. Patients undergoing cardiac surgery with cardiopulmonary bypass and regular PPI medication developed significant more infections retrospectively indicating a clinical impact of the immunosuppressive influence of PPI.

Depressed polymorphonuclear cell functions in periparturient cows that develop postpartum reproductive diseases.

The study was planned to see if there is any important and significant changes in the PMN function in cows suffering from postpartum reproductive diseases (PRD). Blood sampling was done from 41 pregnant cows on 15days prepartum (-15d), calving day (0d), 15days (15d) and 30days (30d) postpartum and thorough gynaecological examination was performed on 0d, 15d, 30d and 45d for diagnosis of PRD like retained placenta (RP), clinical metritis (CM), clinical endometritis (CE) and delayed involution of uterus (DIU). The heparinised blood was used for isolation of PMN leukocytes for estimation of superoxide (SO), hydrogen peroxide (H2O2) and enzyme myeloperoxidase (MPO) activity in each group of cows. The SO production (ΔOD) was greater for normal (0.19±0.05) than cows suffering from RP (-0.12±0.09), CM (-0.15±0.13) and CE (-0.07±0.05) at -15d. The mean value was greater for normal cows (0.12) than the cows with PRD (0.05 to 0.9) at 30d. The H2O2 production was greater for normal than cows with PRD at all sampling days and significantly greater than cows with RP and CE at 15d (p<0.01) and 30d (P<0.05). The MPO activity (μmol/1×107) was greater for normal (18.77±1.27) than for RP (12.52±2.57) and CM (11.31±3.30) cows on 0d. The depressed capability of the PMN from the cows with PRD to produce SO, H2O2 and MPO during the periparturient period indicated their association with the development of RP, CM and CE.

A commercial immune modulating feed additive restores L-selectin and CCL5 expression following dexamethasone treatment of murine immune cells in a MyD88-dependent manner.

Bovine mastitis costs the dairy industry billions of dollars every year and presents a health challenge in dairy facilities. Immunosuppressive effects of the periparturient period increase the incidence of mastitis. During this time, cattle experience an elevation in circulating cortisol, which reduces polymorphonuclear cell function and ability to clear infection. OmniGen-AF (OMN; Phibro Animal Health, Teaneck, NJ) is an immunomodulatory feed additive that alters gene expression and is used to reduce rates of mastitis. We hypothesized that OMN restores gene expression during periods of immune stress through inhibiting the suppressive effects of glucocorticoid receptor signaling on Toll-like receptor signaling. To test our hypothesis, wild-type (WT) or MyD88 knockout mice were supplemented with OMN and challenged with lipopolysaccharide following dexamethasone (Dex) treatment. Polymorphonuclear cell and macrophage RNA was isolated from intraperitoneal lavages and analyzed for gene expression profiles. Treatment of mice with Dex suppressed expression of l-selectin and CCL5 as compared with phosphate-buffered saline treatment of WT mice. Expression of l-selectin and CCL5 was significantly reduced with Dex treatment in control-fed but not OMN-supplemented WT mice. The protective effect of OMN supplementation on l-selectin expression during Dex treatment was abolished in MyD88 knockout mice. These results suggest that OMN supplementation restores responses of certain genes suppressed by Dex in immune cells in a MyD88-dependent manner. Future research will determine the specific Toll-like receptors, transcription factors, and biochemical properties of OMN that restore gene expression in immunosuppressed cells.

From obesity through immunity to type 2 diabetes mellitus

Diabetes mellitus is a metabolic disorder that leads to the development of a number of complications. The etiology of each metabolic complication is undoubtedly multifactorial. Patients with diabetes have increased susceptibility to and severity of infections. The course of infections is also more complicated in the patient group. One of the possible causes of this increased prevalence of infections is defect in immunity. Different disturbances in humoral innate immunity have been described in patients with diabetes. Concerning cellular innate immunity, most studies show decreased functions of polymorphonuclear cells and monocyte/macrophages of these patients compared to cells of healthy subjects. Several studies have shown alterations in peripheral blood mononuclear cells from patients with type 2 diabetes, an effect that contributes to the high incidence of infections in these patients. The gut microbiota plays different roles such as the following: protects against pathogens, helps in the maturation of the immune system, regulates the intestinal hormone secretion, synthesizes vitamin K and several vitamins B, and produces short-chain fatty acids (SCFAs). It also plays a role in immunomodulation and might contribute to the alterations in glucose metabolism. In the present review, I focused on the role of obesity, the immune system, and the gut microbiota in the pathogenesis of type 2 diabetes mellitus, and as a second point, how type 2 diabetes impairs the immune system.

Minocycline affects human neutrophil respiratory burst and transendothelial migration.

This study aimed at investigating the in vitro activity of minocycline and doxycycline on human polymorphonuclear (h-PMN) cell function. h-PMNs were isolated from whole venous blood of healthy subjects; PMN oxidative burst was measured by monitoring ROS-induced oxidation of luminol and transendothelial migration was studied by measuring PMN migration through a monolayer of human umbilical vein endothelial cells. Differences between multiple groups were determined by ANOVA followed by Tukey's multiple comparison test; Student's t test for unpaired data for two groups. Minocycline (1-300µM) concentration dependently and significantly inhibited oxidative burst of h-PMNs stimulated with 100nM fMLP. Ten micromolar concentrations, which are superimposable to C max following a standard oral dose of minocycline, promoted a 29.8±4% inhibition of respiratory burst (P<0.001; n=6). Doxycycline inhibited ROS production with a lesser extent and at higher concentrations. 10-100µM minocycline impaired PMN transendothelial migration, with maximal effect at 100µM (42.5±7%, inhibition, n=5, P<0.001). These results added new insight into anti-inflammatory effects of minocycline exerted on innate immune h-PMN cell function.

Evaluation of immunomodulatory effect of recombinant human granulocyte-macrophage colony-stimulating factor on polymorphonuclear cell from dogs with cancer in vitro.

The objective of this in vitro study was to evaluate the immunomodulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on polymorphonuclear cell (PMN) function in dogs with cancer. PMNs were harvested from dogs with naturally developing cancer as a pre-clinical model to evaluate the immunomodulatory effects of rhGM-CSF on PMN phagocytic and cytotoxic functions, cytokine production and receptor expression. Some aspects of cancer-related PMN dysfunction in dogs with cancer were restored following incubation with rhGM-CSF including PMN phagocytosis, respiratory burst and LPS-induced TNF-α production. In addition, rhGM-CSF increased surface HLA-DR expression on the PMNs of dogs with cancer. These data suggests that dysfunction of innate immune response in dogs with cancer may be improved by rhGM-CSF. The results of this study provided a pathophysiologic rationale for the initiation of clinical trials to continue evaluating rhGM-CSF as an immunomodulatory therapy in dogs with cancer.

A Complement-Optimized EGFR Antibody Improves Cytotoxic Functions of Polymorphonuclear Cells against Tumor Cells.

Complement-dependent cytotoxicity (CDC) has been suggested to be an important mechanism of action of tumor-targeting Abs. However, single unmodified epidermal growth factor receptor (EGFR)-targeting IgG1 Abs fail to trigger efficient CDC. For the current study, we generated a CDC-optimized variant of the EGFR Ab matuzumab (H425 wt) by introducing amino acid substitutions K326A/E333A (H425 mt). This Ab was then used to elucidate the impact of complement activation on the capacity of effector cells such as mononuclear cells (MNC) and polymorphonuclear cells (PMN) to exert Ab-dependent cell-mediated cytotoxicity (ADCC). H425 mt, but not H425 wt, significantly induced complement deposition, release of anaphylatoxins, and CDC against distinct tumor cell lines, whereas no differences in ADCC by MNC or PMN were detected. Notably, stronger cytotoxicity was induced by H425 mt than by H425 wt in whole blood assays and in experiments in which MNC or PMN were combined with serum. Although MNC-ADCC was not affected by C5 cleavage, the cytotoxic activity of PMN in the presence of serum strongly depended on C5 cleavage, pointing to a direct interaction between complement and PMN. Strong cell surface expression of C5a receptors was detected on PMN, whereas NK cells completely lacked expression. Stimulation of PMN with C5a led to upregulation of activated complement receptor 3, resulting in enhanced complement receptor 3-dependent PMN-ADCC against tumor cells. In conclusion, complement-optimized EGFR Abs may constitute a promising strategy to improve tumor cell killing by enhancing the interaction between humoral and cellular effector functions in Ab-based tumor therapy.