A polyoxyethylene sorbitan oleate modified hollow gold nanoparticle system to escape macrophage phagocytosis designed for triple combination lung cancer therapy via LDL-R mediated endocytosis.

Presently, a combination of chemotherapy, radiotherapy, thermotherapy, and other treatments has become a hot topic of research for the treatment of cancer, especially lung cancer. In this study, novel hollow gold nanoparticles (HGNPs) were used as drug carriers, and in order to improve the targeting ability of HGNPs to a lung tumor site, polyoxyethylene sorbitol oleate (PSO) was chosen here as a target ligand since it can be specifically recognized by the low-density lipoprotein (LDL) receptor which is usually over expressed on A549 lung cancer cells. In this way, a PSO-modified doxorubicin-loaded HGNP drug delivery system (PSO-HGNPs-DOX) was constructed and its physicochemical properties, photothermal conversion ability, and drug release of PSO-HGNPs-DOX was investigated. Further, the effects of triple combination therapy, the intracellular uptake, and the ability to escape macrophage phagocytosis of PSO-HGNPs-DOX were also studied using A549 cells in vitro. In addition, an in vivo mouse model was also used to study the targeting of PSO-HGNPs-DOX to lung cancer. PSO-HGNPs-DOX demonstrated a good triple therapeutic effect for lung cancer (A549 cell viability was only 10% at 500 µM) by LDL receptor mediated endocytosis and was able to escape macrophage phagocytosis to enhance its accumulation at the target site. Therefore, PSO-HGNPs-DOX is a novel, safe, promising, and targeted drug carrier designed for triple combination lung cancer therapy which should be further studied for such applications.

Direct and high-throughput assays for human cell killing through trogocytosis by Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebiasis. Pathogenesis is associated with profound damage to human tissues. We previously showed that amoebae kill human cells through trogocytosis. Trogocytosis is likely to underlie tissue damage during infection, although the mechanism is still unknown. Trogocytosis is difficult to assay quantitatively, which makes it difficult to study. Here, we developed two new, complementary assays to measure trogocytosis by quantifying human cell death. One assay uses CellTiterGlo, a luminescent readout for ATP, as a proxy for cell death. We found that the CellTiterGlo could be used to detect death of human cells after co-incubation with amoebae, and that it was sensitive to inhibition of actin or the amoeba surface Gal/GalNAc lectin, two conditions that are known to inhibit amoebic trogocytosis. The other assay uses two fluorescent nuclear stains to directly differentiate live and dead human cells by microscopy, and is also sensitive to inhibition of amoebic trogocytosis through interference with actin. Both assays are simple and inexpensive, can be used with suspension and adherent human cell types, and are amenable to high-throughput approaches. These new assays are tools to improve understanding of trogocytosis and amoebiasis pathogenesis.

Large-Scale Production of Human iPSC-Derived Macrophages for Drug Screening.

Tissue-resident macrophages are key players in inflammatory processes, and their activation and functionality are crucial in health and disease. Numerous diseases are associated with alterations in homeostasis or dysregulation of the innate immune system, including allergic reactions, autoimmune diseases, and cancer. Macrophages are a prime target for drug discovery due to their major regulatory role in health and disease. Currently, the main sources of macrophages used for therapeutic compound screening are primary cells isolated from blood or tissue or immortalized or neoplastic cell lines (e.g., THP-1). Here, we describe an improved method to employ induced pluripotent stem cells (iPSCs) for the high-yield, large-scale production of cells resembling tissue-resident macrophages. For this, iPSC-derived macrophage-like cells are thoroughly characterized to confirm their cell identity and thus their suitability for drug screening purposes. These iPSC-derived macrophages show strong cellular identity with primary macrophages and recapitulate key functional characteristics, including cytokine release, phagocytosis, and chemotaxis. Furthermore, we demonstrate that genetic modifications can be readily introduced at the macrophage-like progenitor stage in order to interrogate drug target-relevant pathways. In summary, this novel method overcomes previous shortcomings with primary and leukemic cells and facilitates large-scale production of genetically modified iPSC-derived macrophages for drug screening applications.

Long-Tailed Unconventional Class I Myosins in Health and Disease.

Long-tailed unconventional class I myosin, Myosin 1E (MYO1E) and Myosin 1F (MYO1F) are motor proteins that use chemical energy from the hydrolysis of adenosine triphosphate (ATP) to produce mechanical work along the actin cytoskeleton. On the basis of their motor properties and structural features, myosins perform a variety of essential roles in physiological processes such as endocytosis, exocytosis, cell adhesion, and migration. The long tailed unconventional class I myosins are characterized by having a conserved motor head domain, which binds actin and hydrolyzes ATP, followed by a short neck with an isoleucine-glutamine (IQ) motif, which binds calmodulin and is sensitive to calcium, and a tail that contains a pleckstrin homology domain (PH), a tail homology 1 domain (TH1), wherein these domains allow membrane binding, a tail homology 2 domain (TH2), an ATP-insensitive actin-binding site domain, and a single Src homology 3 domain (SH3) susceptible to binding proline rich regions in other proteins. Therefore, these motor proteins are able to bind actin, plasma membrane, and other molecules (adaptor, kinases, membrane proteins) that contribute to their function, ranging from increasing membrane tension to molecular trafficking and cellular adhesion. MYO1E and MYO1F function in host self-defense, with a better defined role in innate immunity in cell migration and phagocytosis. Impairments of their function have been identified in patients suffering pathologies ranging from tumoral processes to kidney diseases. In this review, we summarize our current knowledge of specific features and functions of MYO1E and MYO1F in various tissues, as well as their involvement in disease.

Role of dietary fatty acids in microglial polarization in Alzheimer's disease.

Microglial polarization is an utmost important phenomenon in Alzheimer's disease that influences the brain environment. Polarization depends upon the types of responses that cells undergo, and it is characterized by receptors present on the cell surface and the secreted cytokines to the most. The expression of receptors on the surface is majorly influenced by internal and external factors such as dietary lipids. Types of fatty acids consumed through diet influence the brain environment and glial cell phenotype and types of receptors on microglia. Reports suggest that dietary habits influence microglial polarization and the switching of microglial phenotype is very important in neurodegenerative diseases. Omega-3 fatty acids have more influence on the brain, and they are found to regulate the inflammatory stage of microglia by fine-tuning the number of receptors expressed on microglia cells. In Alzheimer's disease, one of the pathological proteins involved is Tau protein, and microtubule-associated protein upon abnormal phosphorylation detaches from the microtubule and forms insoluble aggregates. Aggregated proteins have a tendency to propagate within the neurons and also become one of the causes of neuroinflammation. We hypothesize that tuning microglia towards anti-inflammatory phenotype would reduce the propagation of Tau in Alzheimer's disease.

Vitamin-D3 (α-1, 25(OH) 2D3) Protects Retinal Pigment Epithelium From Hyperoxic Insults.

Purpose: Oxidative stress affects the retinal pigment epithelium (RPE) leading to development of vascular eye diseases. Cholecalciferol (VIT-D) is a known modulator of oxidative stress and angiogenesis. This in vitro study was carried out to evaluate the protective role of VIT-D on RPE cells incubated under hyperoxic conditions. Methods: Cadaver primary RPE (PRPE) cells were cultured in hyperoxia (40% O2) with or without VIT-D (α-1, 25(OH) 2D3). The functional and physiological effects of PRPE cells with VIT-D treatment were analyzed using molecular and biochemical tools. Results: Vascular signaling modulators, such as vascular endothelial growth factor (VEGF) and Notch, were reduced in hyperoxic conditions but significantly upregulated in the presence of VIT-D. Additionally, PRPE conditioned medium with VIT-D induced the tubulogenesis in primary human umbilical vein endothelial cells (HUVEC) cells. VIT-D supplementation restored phagocytosis and transmembrane potential in PRPE cells cultured under hyperoxia. Conclusions: VIT-D protects RPE cells and promotes angiogenesis under hyperoxic insult. These findings may give impetus to the potential of VIT-D as a therapeutic agent in hyperoxia induced retinal vascular diseases.

DHA Sensor GPR120 in Host Defense Exhibits the Dual Characteristics of Regulating Dendritic Cell Function and Skewing the Balance of Th17/Tregs.

In addition to functioning as an antioxidant, anti-inflammatory and age-defying cellular component, DHA impacts the immune system by facilitating the pathogen invasion. The mechanism through which DHA regulates immune suppression remains obscure. In our study, we postulated that DHA might interact with GPR120 to shape the dendritic cell (DC) differentiation and subsequently drive T cell proliferation during the virus infection. In vitro, the proportion of costimulatory molecules and HLA-DR on DC that generated from exogenous and endogenous (fad3b expression) DHA supplemented mice were significantly lower than wild-type mice. Given the importance of FAs, DHA is not only a critical cellular constituent but also a cell signaling molecule and FA deficiency reduces DC generation; we used GPR120-/- mice to determine whether DHA receptor deficiency disorders DC maturation processing. Novelty, the expression of GPR120 on DC from wild-type (WT) mice was inversely related to DC activation and DC from the GPR120-/- mice maintained a spontaneous maturation status. In vivo, both the excessive activation of GPR120 by DHA and the deletion of GPR120 effectively skewed the balance of Th17/Tregs and reduced the production of VNA and protection of vaccination. Overall, our results revealed a mechanism that the GPR120 self-regulation plays a crucial role in sensing DHA variation, which provides a new prospect for therapeutic manipulation in autoimmune diseases and the design of a vaccine adjuvant.

Melatonin Action on the Activity of Phagocytes from the Colostrum of Obese Women.

BACKGROUND AND OBJECTIVES: Breastfeeding promotion is an important public health strategy for counter-balancing the negative effects of maternal overweight and obesity. Colostrum contains melatonin, which can attenuate the impacts of excessive maternal weight and boost the infant's immune system. Therefore, the objective of this study was to analyze the effects of melatonin on mononuclear (MN) phagocytes from the colostrum of women with pre-gestational obesity. Materials and Methods: Colostrum samples were collected postpartum from 100 women at a public hospital in São Paulo, Brazil. The donors were divided into two groups: the control group and the high body mass index (BMI) group. Melatonin levels in the colostrum were determined by an ELISA Kit, and the functional activity of MN cells was assessed using the phagocytosis assay by flow cytometry, and reactive oxygen species (ROS), intracellular calcium, and apoptosis were assessed by fluorimetry using a microplate reader. RESULTS: The colostrum of mothers with pre-gestational high BMI exhibited higher melatonin levels (p < 0.05) and lower phagocytosis (p < 0.05) and ROS release (p < 0.05). Superoxide release was similar between the normal and high BMI groups (p > 0.05). Intracellular calcium release and apoptosis were also higher in the high BMI group (p < 0.05). Melatonin levels likely increased the phagocytosis rate and reduced intracellular calcium release and the apoptosis index (p < 0.05). CONCLUSIONS: The results suggest that melatonin is a possible mechanism for maternal-infant protection against obesity and restores the functional activity of colostrum phagocytes in obese mothers.

Sirt1 ameliorates monosodium urate crystal-induced inflammation by altering macrophage polarization via the PI3K/Akt/STAT6 pathway.

OBJECTIVES: Acute gout is an inflammatory response to MSU crystals. In our previous research, Sirt1 was shown to have an effect in preventing acute gouty inflammation. In the current study, we aimed to investigate the underlying mechanism involving Sirt1 in acute gout. METHODS: The cytological changes and Sirt1 expression in the synovium were observed in patients with acute or intermittent gout. The effect of Sirt1 and its mechanism in gout were studied in macrophages, C57BL/6 mice and Sirt1+/- mice. RESULTS: Sirt1 expression was increased in the peripheral blood mononuclear cells (PBMCs) of patients with acute gout but not in the chronic tophus tissue. The arthritis score and numbers of inflammatory cells in injured paw tissue from murine gout models were upregulated in Sirt1+/- mice compared with wild-type mice. A PCR array of the paw tissue from murine gout models indicated that Sirt1 activation might attenuate MSU-induced inflammation by altering the polarization state of macrophages. Furthermore, in patients with acute gout, the phagocytosis of MSU crystals by a macrophage was found in a smear of the joint fluid and large amounts of macrophages were also found in the synovium. The activation of Sirt1 in gouty mice actually decreased the tendency toward M1 polarization. The inhibition of PI3K/Akt partially blocked the anti-inflammatory effect of Sirt1 and the translocation of STAT6, and phosphorylated STAT6 expression was decreased in RAW 264.7 cells treated with MSU crystals. CONCLUSION: Our studies revealed that Sirt1 ameliorates MSU-induced inflammation by altering macrophage polarization via the PI3K/Akt/STAT6 pathway.

Selenoprotein SELENOK Enhances the Migration and Phagocytosis of Microglial Cells by Increasing the Cytosolic Free Ca2+ Level Resulted from the Up-Regulation of IP3R.

Enhancing the migration and phagocytosis of microglial cells is of great significance for the reducing of the risk of the neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The effect of mouse selenoprotein K (mSELENOK) on the migration and phagocytosis of BV2 microglial cells and its mechanism were studied. The results showed that the over-expression of mSELENOK can increase the migratory and phagocytic abilities of the microglial cells, while the knockdown of mSELENOK can decrease the migratory and phagocytic abilities of the cells. The cytosolic free Ca2+ level and inositol trisphosphate receptor (IP3R) mRNA transcript and protein expression were also increased significantly as the consequence of the over-expression of mSELENOK in the microglial cells. On the contrary, the level of cytosolic free Ca2+ and the mRNA transcript and protein expression of IP3R in mSELENOK knockdown cells were decreased significantly. 2-aminoethoxydiphenyl borate (2-APB), an antagonist of IP3R, could prevent the increased migration, phagocytosis, and cytosolic free Ca2+ level of mSELENOK over-expressed microglial cells, and knockdown of IP3R3 could reduce the increased cytosolic Ca2+ level in mSELENOK over-expressed microglial cells. Further studies revealed that selenium supplement (Na2SeO3) can increase the expression of mSELENOK in microglial cells significantly. In summary, these data suggest that mSELENOK can increase cytosolic free Ca2+ level of microglial cells by up-regulating the expression of IP3R, thus enhancing the migration and phagocytosis of microglial cells. Our results indicated that mSELENOK is an important selenoprotein, which plays a role in trace element selenium's functions and can enhance the migration and phagocytosis of microglial cells.

Immunomodulatory Activities of Polysaccharides from White Button Mushroom, Agaricus bisporus (Agaricomycetes), Fruiting Bodies and Cultured Mycelia in Healthy and Immunosuppressed Mice.

Agaricus bisporus is a very important edible and medicinal mushroom. In this study, we systematically investigated the monosaccharide composition, methylation, and immunomodulatory activities of polysaccharides from A. bisporus fruiting bodies (FPS), cultured mycelia (IPS), and fermentation broth (EPS). The results indicated that FPS was mainly composed of mannose; IPS, of glucose; and EPS, of galactose. However, the methylation results indicated that FPS, IPS, and EPS possessed different polysaccharide structures. Furthermore, FPS, IPS, and EPS caused remarkable increases in the thymus and spleen indexes; in the amounts of serum cytokines containing interleukin (IL)-2, IL-4, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ); in the counts of CD3+CD4+ lymphocytes and the ratio of CD4+ to CD8+ T lymphocytes; however, they decreased the counts of CD3+CD8+ lymphocytes in normal mice. Finally, in cyclophosphamide-treated mice, the FPS, IPS, and EPS were able to significantly restore the thymus and spleen indexes, lymphocyte proliferation, phagocytotic activity of peritoneal macrophages, and levels of IL-2, IL-6, IL-10, IL-17, TNF-α, and immunoglobin G. These findings suggest that FPS, IPS, and EPS could all be exploited as immunomodulatory agents and potential immunotherapeutic medicines for patients with inadequate immune function.

Effect of Cytokines and Hyperthermia on Phagocytosis and Phosphatidylserine Externalization: Implication for the Pathophysiology of Hemophagocytic Syndrome.

OBJECTIVE: An aberrant production of inflammatory cytokines, with resultant febrile response, is a cardinal finding of hemophagocytic syndrome. A role of inflammatory cytokines on phagocytosis and clearance of apoptotic cells or particles has been shown, but effects of cytokines or hyperthermia on phagocytosis of viable blood cells were not fully understood. We examined effects of cytokines and hyperthermia on phagocytosis, and externalization of phosphatidylserine on the surface of phagocytosed blood cells, to clarify the pathophysiology of hemophagocytic syndrome. METHODS: THP-1 macrophage cells were incubated with non-opsonized and opsonized sheep erythrocytes (SE) in the presence of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), macrophage-colony stimulating factor (M-CSF), interleukin (IL)-6, IL-10 or IL-18, and phagocytic activity was analyzed. Co-operative effect between cytokines was also examined. In addition, SE were incubated at 37 or 39°C, and phagocytic activity was analyzed. After treatment of SE with cytokine or hyperthermia, phosphatidylserine expression of the cell surface was analyzed by detecting Annexin V-positive cells. RESULTS: IL-6, IL-10 and IL-18 significantly increased phagocytosis of non-opsonized SE, but IFN-γ suppressed it. Phagocytosis of opsonized SE was significantly increased by any of the cytokines. IFN-γ suppressed and IL-10 enhanced phagocytic activity induced by other cytokines in non-opsonized SE, while in opsonized SE, both cytokines co-operated with other cytokines to enhance phagocytosis. Incubation of SE at a high temperature (39°C) resulted in increased phagocytic activity, as compared to SE incubated at 37°C. Cytokines and a high temperature did not increase the number of Annexin V-positive SE. CONCLUSIONS: IL-6, IL-10 and IL-18 can augment phagocytosis of viable blood cells, whether cells are opsonized or not. Hyperthermia also enhances phagocytosis. These in vitro data suggest that therapy for targeting cytokine (IL-6, IL-10 or IL-18) by using biologics or small molecule drugs may be beneficial for the treatment of hemophagocytic syndrome. Unlike the case of apoptotic cells, phagocytosis of viable blood cells seems to be mediated via phosphatidylserine-independent manner.

The serrulatane diterpenoid natural products RAD288 and RAD289 stimulate properties of olfactory ensheathing cells useful for neural repair therapies.

Olfactory ensheathing cells (OECs) are being trialled for cell transplantation therapies for neural repair as they have unique properties which can enhance neuron regeneration. However, improvements in cell viability, proliferation and migration are needed to enhance therapeutic outcomes. Growth factors can enhance cell activity, but they can also induce side effects as they can act on numerous cell types. An alternative approach is to identify natural products (NPs) that more selectively activate specific cell functions. We have examined two pure NPs, 3-acetoxy-7,8-dihydroxyserrulat-14-en-19-oic acid (RAD288) and 3,7,8-trihydroxyserrulat-14-en-19-oic acid (RAD289) isolated from the Australian plant Eremophila microtheca. We determined that RAD288 and RAD289 stimulated the viability and proliferation of OECs in two-dimensional cultures and increased cell viability in three-dimensional spheroids. Both compounds also enhanced OEC-mediated phagocytosis of neural debris. However, only RAD288 stimulated migration of OECs, demonstrating that key structural changes to the compound can dramatically affect the resultant cellular action. In addition, cell-type specific action is highlighted by the result that neither compound stimulated the viability of Schwann cells which are a closely-related glial cell type. Therefore, these small molecules may have high potential for selective activation of specific therapeutically-useful activities of OECs for transplantation therapies to repair the nervous system.

Effects of energy supply and nicotinic acid supplementation on phagocytosis and ROS production of blood immune cells of periparturient primi- and pluriparous dairy cows.

Effects of energy supply and nicotinic acid (NA) supplementation on the phagocytic activity of polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC), and on ROS production in PMN of periparturient cows differing in parity were examined. 29 pluriparous and 18 primiparous cows were allocated to four different feeding groups from 42days prepartum until 100days postpartum. They were fed either a ration with a low concentrate proportion of 30% (LC) or a high concentrate proportion of 60% (HC). After parturition all animals received 30% concentrate which was increased to 50% either within 16 (LC) or within 24days (HC). The different concentrate feeding strategies aimed at triggering differences in postpartum lipolysis. Half of the animals per group were supplemented with 24g per day of NA from 42days prepartum until 24days postpartum. All investigated parameters varied significantly over time compared to parturition (p<0.05). Numbers of phagocytosing PMN and PBMC increased in the course of the experiment, whereas the amount of engulfed bacteria per cell decreased between 42 and 11days prepartum. Percentage of basal ROS producing PMN decreased strongly before parturition and reached initial values only at 28days in milk again. Mean fluorescence intensity (MFI) in these ROS producing cells, however, increased before parturition. Oxidative burst stimulation in PMN was reduced around parturition but the amount of ROS produced in the stimulated cells was increased. Pluriparous cows exhibited higher numbers of basal ROS producing PMN and phagocytic PBMC. NA supplementation influenced phagocytosis in blood leukocytes.

Neutrophil phagocytosis and respiratory burst activity of dairy cows during the transition period and early lactation.

INTRODUCTION: Hormonal and metabolic changes, as well as energy imbalance, can affect health, production and reproductive performance of dairy cows. In the present study, we evaluated phagocytosis and respiratory burst neutrophil activity during the transition period and early lactation and compared it with biochemical and hematological parameters in dairy cows. METHODOLOGY: Simmental cows (n = 21) were enrolled in the study. Whole blood samples were collected weekly from 3 weeks pre- calving until 6 weeks post calving. Basic metabolic and blood parameters were assessed by routine laboratory analyses, while neutrophil functions were analyzed by commercial test kits. RESULTS: Optimal neutrophil response was observed pre and post calving. The highest value was recorded in the 6th week after calving (89.54 ± 7.61%) and being significantly higher (p < 0.01) as compared to values recorded at two and one week before and one week after calving. The percentage of activated neutrophils was high during the entire study period: from 70.80 ± 5.22% at the beginning of the study to 89.54 ± 7.61% at the end of the study. During the study period, production of Reactive Oxidative Species by neutrophils was positively correlated with ß-hydroxybutyrat and non-esterified fatty acids values (0.454** and 0.423**, respectively) and calcium levels (0.164* and 0.212**, respectively). CONCLUSIONS: The most prominent changes in all parameters had no influence on phagocytic and respiratory burst activity of neutrophils. Neutrophil function is preserved at the optimal level during the transition period and early lactation in Simmental cows.

Phagocytosis Deficiency of Macrophages in NOD.H-2h4 Mice Accelerates the Severity of Iodine-Induced Autoimmune Thyroiditis.

Apoptosis occurs in many autoimmune diseases. Excess iodine induces thyrocyte apoptosis and increases the incidence and prevalence of autoimmune thyroiditis (AIT). However, the sequence of events between the appearance of thyrocyte apoptosis and the occurrence of thyroiditis remains uncharacterized. Furthermore, few studies have investigated the role of macrophage phagocytosis in the development of AIT. Therefore, we evaluated the relationship between apoptosis and inflammatory infiltration in NOD.H-2h4 mouse thyroids by comparing the sequence of events in tissue samples. We also investigated the role of macrophages by comparing macrophage phagocytosis function in BALB/c, C57BL/6, and NOD.H-2h4 mice treated with different levels of iodine. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and thyroid inflammatory scores revealed that apoptosis (2 weeks) occurred before inflammatory infiltration (4 weeks). Phosphatidylserine (PS) expression on the extracellular surface of the cell membrane and double-stranded DNA fragments associated with apoptosis appeared at 2 and 8 weeks, respectively. Additionally, although apoptosis was enhanced in the thyroids of mice supplemented with excess iodine (0.05 ± 0.12 vs 1.63 ± 0.82% for BALB/c, 0.09 ± 0.14 vs 1.51 ± 0.34% for C57BL/6, and 0.07 ± 1.11 vs 4.72 ± 0.62% for NOD.H-2h4 mice), only NOD.H-2h4 mouse thyroids presented with inflammation. Furthermore, macrophages from NOD.H-2h4 mice (44.46 ± 1.79%) exhibited decreased phagocytotic activity relative to that in BALB/c (54.21 ± 4.58%) and C57BL/6 (58.96 ± 4.04%) mice. There were no differences in phagocytosis function between NOD.H-2h4 mice supplemented with excess iodine or left untreated (24.50 ± 2.66 vs 21.71 ± 1.79%, p = 0.06). In conclusion, deficiencies in the apoptosis clearance of macrophages in NOD.H-2h4 mice may constitute an early pathogenic mechanism in AIT that is not influenced by iodine intake.

Impaired microglia process dynamics post-stroke are specific to sites of secondary neurodegeneration.

Stroke induces tissue death both at the site of infarction and at secondary sites connected to the primary infarction. This latter process has been referred to as secondary neurodegeneration (SND). Using predominantly fixed tissue analyses, microglia have been implicated in regulating the initial response at both damage sites post-stroke. In this study, we used acute slice based multiphoton imaging, to investigate microglia dynamic process movement in mice 14 days after a photothrombotic stroke. We evaluated the baseline motility and process responses to locally induced laser damage in both the peri-infarct (PI) territory and the ipsilateral thalamus, a major site of post-stroke SND. Our findings show that microglia process extension toward laser damage within the thalamus is lost, yet remains robustly intact within the PI territory. However, microglia at both sites displayed an activated morphology and elevated levels of commonly used activation markers (CD68, CD11b), indicating that the standardly used fixed tissue metrics of microglial "activity" are not necessarily predictive of microglia function. Analysis of the purinergic P2 Y12 receptor, a key regulator of microglia process extension, revealed an increased somal localization on nonresponsive microglia in the thalamus. To our knowledge, this is the first study to identify a non-responsive microglia phenotype specific to areas of SND post-stroke, which cannot be identified by the classical assessment of microglia activation but rather the localization of P2 Y12 to the soma.

Oxidative stress induced by Se-deficient high-energy diet implicates neutrophil dysfunction via Nrf2 pathway suppression in swine.

The mechanism of the interaction between Se deficiency and high energy remains limited. The aim of the current study was to identify whether Se-deficient, high-energy diet can induce oxidative stress, and downregulate the Nrf2 pathway and phagocytic dysfunction of neutrophils. We detected the phagocytic activity, ROS production, protein levels of Nrf2 and Nrf2 downstream target genes, and the mRNA levels of 25 selenoproteins, heat shock proteins, and cytokines in neutrophils. Cytokine ELISA kits were used to measure the serum cytokines. The concentration of ROS was elevated (P < 0.05) in obese swine fed on a low Se diet (less than 0.03 mg/kg Se) compared to control swine. The protein levels of Nrf2 and its downstream target genes were depressed during Se deficiency and high-energy intake. The mRNA levels of 16 selenoproteins were significantly decreased (P < 0.05) in the Se-deficient group and Se-deficient, high-energy group compared to the control group. However, the mRNA levels of 13 selenoproteins in peripheral blood neutrophils were upregulated in high energy group, except TrxR1, SelI and SepW. In summary, these data indicated that a Se-deficient, high-energy diet inhibits the Nrf2 pathway and its regulation of oxidative stress, and prompted a pleiotropic mechanism that suppresses phagocytosis.

Flowers of Clerodendrum volubile exacerbate immunomodulation by suppressing phagocytic oxidative burst and modulation of COX-2 activity.

The immunomodulatory potentials of the crude methanolic extract and fractions [n-hexane (Hex), n-dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH)] of Clerodendrum volubile flowers were investigated on whole blood phagocytic oxidative burst using luminol-amplified chemiluminescence technique. They were also investigated for their free radicals scavenging activities. The DCM fraction showed significant (p<0.05) anti-oxidative burst and free radical scavenging activities indicating high immunomodulatory and antioxidant potencies respectively. Cytotoxicity assay of the DCM fraction revealed a cytotoxic effect on CC-1 normal cell line. GCMS analysis revealed the presence of triacetin; 3,6-dimethyl-3-octanol; 2R - Acetoxymethyl-1,3,3-trimethtyl - 4t - (3-methyl-2-buten-1-yl) - 1c - cyclohexanol and Stigmastan - 3,5-diene in DCM fraction. These compounds were docked with the active sites of cyclooxygenase-2 (COX-2). Triacetin, 3,6-dimethyl-3-Octanol, and 2R-Acetoxymethyl-1,3,3-trimethtyl-4t-(3-methyl-2-buten-1-yl)-1c-cyclohexanol docked comfortably with COX-2 with good scoring function (-CDocker energy) indicating their inhibitory potency against COX-2. 3,6-dimethyl-3-Octanol, displayed the lowest predicted free energy of binding (-21.4kcalmol-1) suggesting its stronger interaction with COX-2, this was followed by 2R - Acetoxymethyl-1, 3, 3-trimethtyl-4t-(3-methyl-2-buten-1-yl)-1c-cyclhexanol (BE=-20.5kcalmol-1), and triacetin (BE=-10.9kcalmol-1). Stigmastan - 3,5-diene failed to dock with COX-2. The observed suppressive effect of the DCM fraction of C. volubile flower methanolic extract on phagocytic oxidative burst indicates an immunomodulatory potential. This is further reflected in its free scavenging activities and synergetic modulation of COX-2 activities by its identified compounds in silico.

Deletion of calponin 2 in macrophages attenuates the severity of inflammatory arthritis in mice.

Calponin is an actin cytoskeleton-associated protein that regulates motility-based cellular functions. Three isoforms of calponin are present in vertebrates, among which calponin 2 encoded by the Cnn2 gene is expressed in multiple types of cells, including blood cells from the myeloid lineage. Our previous studies demonstrated that macrophages from Cnn2 knockout (KO) mice exhibit increased migration and phagocytosis. Intrigued by an observation that monocytes and macrophages from patients with rheumatoid arthritis had increased calponin 2, we investigated anti-glucose-6-phosphate isomerase serum-induced arthritis in Cnn2-KO mice for the effect of calponin 2 deletion on the pathogenesis and pathology of inflammatory arthritis. The results showed that the development of arthritis was attenuated in systemic Cnn2-KO mice with significantly reduced inflammation and bone erosion than that in age- and stain background-matched C57BL/6 wild-type mice. In vitro differentiation of calponin 2-null mouse bone marrow cells produced fewer osteoclasts with decreased bone resorption. The attenuation of inflammatory arthritis was confirmed in conditional myeloid cell-specific Cnn2-KO mice. The increased phagocytotic activity of calponin 2-null macrophages may facilitate the clearance of autoimmune complexes and the resolution of inflammation, whereas the decreased substrate adhesion may reduce osteoclastogenesis and bone resorption. The data suggest that calponin 2 regulation of cytoskeleton function plays a novel role in the pathogenesis of inflammatory arthritis, implicating a potentially therapeutic target.