Modulation of polymorphonuclear neutrophil functions by astrocytes

<p>Abstract</p> <p>Background</p> <p>Neuroinflammation is a complex process involving cells from the immune system and the central nerve system (CNS). Polymorphonuclear neutrophils (PMN) are the most abundant class of white blood cells, and typically the first type of leukocyte recruited to sites of inflammation. In the CNS, astrocytes are the most abundant glial cell population and participate in the local innate immune response triggered by a variety of insults. In the present study, we investigated the impacts of astrocytes on PMN function.</p> <p>Methods</p> <p>Primary astrocyte cultures were derived from postnatal C57BL/6 mice and primary neutrophils were isolated from 8 to 12 weeks old C57BL/6 mice. PMNs respiratory burst was analyzed by H2DCFDA assay. For phagocytosis assay, neutrophils were incubated with FITC-labeled E. coli and the phagocytosis of E coli was determined by flow cytometer. PMNs degranulation was determined by myeloperoxidase assay. Cytokine expression was determined by real-time PCR. To determine the involvement of different signaling pathway, protein lysates were prepared and western blots were conducted to assess the activation of Akt, Erk1/2, and p38.</p> <p>Results</p> <p>Using ex vivo neutrophils and primary astrocyte cultures, our study demonstrated that astrocytes differentially regulate neutrophil functions, depending upon whether the interactions between the two cell types are direct or indirect. Upon direct cell-cell contact, astrocytes attenuate neutrophil apoptosis, respiratory bust, and degranulation, while enhancing neutrophil phagocytic capability and pro-inflammatory cytokine expression. Through indirect interaction with neutrophils, astrocytes attenuate apoptosis and enhance necrosis in neutrophils, augment neutrophil phagocytosis and respiratory burst, and inhibit neutrophil degranulation. In addition, astrocytes could augment Akt, Erk1/2, and p38 activation in neutrophils.</p> <p>Conclusions</p> <p>Astrocytes differentially regulate neutrophil functions through direct or indirect interactions between the two cell types. The diversified actions of astrocytes on neutrophils might provide protection against potential microbial infections given compromised blood-brain barrier integrity under certain neuropathological conditions. The complex actions of astrocytes on neutrophils could provide further insight to harness the inflammatory response to promote CNS repair.</p

Sublingual Immunization with Chimeric C1q/CD40 Ligand/HIV Virus-like Particles Induces Strong Mucosal Immune Responses against HIV

Development of a vaccine that can elicit robust HIV specific antibody responses in the mucosal compartments is desired for effective prevention of HIV via sexual transmission. However, the current mucosal vaccines have either poor immunogenicity when administered orally or invite safety concerns when administered intranasally. Sublingual immunization has received more attention in recent years based on its efficiency in inducing systemic and mucosal immune responses in both mucosal and extra-mucosal tissues. To facilitate the transport of the immunogen across the sub-mucosal epithelial barrier, we found that CD91, the receptor of C1q, is prevalently expressed in the sublingual mucosal lining, and thus, a modified chimeric C1q surface conjugated CD40L/HIV VLP was generated. The ability of this chimeric C1q/CD40L/HIV VLP to bind, cross the epithelial layer, access and activate the sub-mucosal layer dendritic cells (DCs), and ultimately induce enhanced mucosal and systemic immune responses against HIV is evaluated in this study. We found that C1q/CD40L/HIV VLPs have enhanced binding, increased transport across the epithelial layer, and upregulate DC activation markers as compared to CD40L/HIV VLPs alone. Mice immunized with C1q/CD40L/HIV VLPs by sublingual administration showed higher levels of IgA salivary antibodies against both HIV Gag and Env than mice immunized with CD40L/HIV VLPs. Moreover, sublingual immunization with C1q/CD40L/HIV VLPs induced more Env- and Gag-specific IFN-γ producing T cells than the CD40L/HIV VLPs group. Interestingly, C1q/CD40L/HIV VLP immunization can also induce more mucosal homing T cells than that in CD40L/HIV VLP group. Our data suggest that incorporation of C1q to CD40L/HIV VLPs is a promising novel strategy and that the sublingual immunization can be a favorite immunization route for HIV mucosal vaccines

Sublingual Immunization with Chimeric C1q/CD40 Ligand/HIV Virus-like Particles Induces Strong Mucosal Immune Responses against HIV

Development of a vaccine that can elicit robust HIV specific antibody responses in the mucosal compartments is desired for effective prevention of HIV via sexual transmission. However, the current mucosal vaccines have either poor immunogenicity when administered orally or invite safety concerns when administered intranasally. Sublingual immunization has received more attention in recent years based on its efficiency in inducing systemic and mucosal immune responses in both mucosal and extra-mucosal tissues. To facilitate the transport of the immunogen across the sub-mucosal epithelial barrier, we found that CD91, the receptor of C1q, is prevalently expressed in the sublingual mucosal lining, and thus, a modified chimeric C1q surface conjugated CD40L/HIV VLP was generated. The ability of this chimeric C1q/CD40L/HIV VLP to bind, cross the epithelial layer, access and activate the sub-mucosal layer dendritic cells (DCs), and ultimately induce enhanced mucosal and systemic immune responses against HIV is evaluated in this study. We found that C1q/CD40L/HIV VLPs have enhanced binding, increased transport across the epithelial layer, and upregulate DC activation markers as compared to CD40L/HIV VLPs alone. Mice immunized with C1q/CD40L/HIV VLPs by sublingual administration showed higher levels of IgA salivary antibodies against both HIV Gag and Env than mice immunized with CD40L/HIV VLPs. Moreover, sublingual immunization with C1q/CD40L/HIV VLPs induced more Env- and Gag-specific IFN-&gamma; producing T cells than the CD40L/HIV VLPs group. Interestingly, C1q/CD40L/HIV VLP immunization can also induce more mucosal homing T cells than that in CD40L/HIV VLP group. Our data suggest that incorporation of C1q to CD40L/HIV VLPs is a promising novel strategy and that the sublingual immunization can be a favorite immunization route for HIV mucosal vaccines

Methylene blue as a cerebral metabolic and hemodynamic enhancer.

By restoring mitochondrial function, methylene blue (MB) is an effective neuroprotectant in many neurological disorders (e.g., Parkinson's and Alzheimer's diseases). MB has also been proposed as a brain metabolic enhancer because of its action on mitochondrial cytochrome c oxidase. We used in vitro and in vivo approaches to determine how MB affects brain metabolism and hemodynamics. For in vitro, we evaluated the effect of MB on brain mitochondrial function, oxygen consumption, and glucose uptake. For in vivo, we applied neuroimaging and intravenous measurements to determine MB's effect on glucose uptake, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO(2)) under normoxic and hypoxic conditions in rats. MB significantly increases mitochondrial complex I-III activity in isolated mitochondria and enhances oxygen consumption and glucose uptake in HT-22 cells. Using positron emission tomography and magnetic resonance imaging (MRI), we observed significant increases in brain glucose uptake, CBF, and CMRO(2) under both normoxic and hypoxic conditions. Further, MRI revealed that MB dramatically increased CBF in the hippocampus and in the cingulate, motor, and frontoparietal cortices, areas of the brain affected by Alzheimer's and Parkinson's diseases. Our results suggest that MB can enhance brain metabolism and hemodynamics, and multimetric neuroimaging systems offer a noninvasive, nondestructive way to evaluate treatment efficacy

Neuroprotective Actions of Methylene Blue and Its Derivatives

<div><p>Methylene blue (MB), the first lead chemical structure of phenothiazine and other derivatives, is commonly used in diagnostic procedures and as a treatment for methemoglobinemia. We have previously demonstrated that MB could function as an alternative mitochondrial electron transfer carrier, enhance cellular oxygen consumption, and provide protection <em>in vitro</em> and in rodent models of Parkinson’s disease and stroke. In the present study, we investigated the structure-activity relationships of MB <em>in vitro</em> using MB and six structurally related compounds. MB reduces mitochondrial superoxide production via alternative electron transfer that bypasses mitochondrial complexes I-III. MB mitigates reactive free radical production and provides neuroprotection in HT-22 cells against glutamate, IAA and rotenone toxicity. Distinctly, MB provides no protection against direct oxidative stress induced by glucose oxidase. Substitution of a side chain at MB’s 10-nitrogen rendered a 1000-fold reduction of the protective potency against glutamate neurototoxicity. Compounds without side chains at positions 3 and 7, chlorophenothiazine and phenothiazine, have distinct redox potentials compared to MB and are incapable of enhancing mitochondrial electron transfer, while obtaining direct antioxidant actions against glutamate, IAA, and rotenone insults. Chlorophenothiazine exhibited direct antioxidant actions in mitochondria lysate assay compared to MB, which required reduction by NADH and mitochondria. MB increased complex IV expression and activity, while 2-chlorphenothiazine had no effect. Our study indicated that MB could attenuate superoxide production by functioning as an alternative mitochondrial electron transfer carrier and as a regenerable anti-oxidant in mitochondria.</p> </div

Methylene Blue as a Cerebral Metabolic and

By restoring mitochondrial function, methylene blue (MB) is an effective neuroprotectant in many neurological disorders (e.g., Parkinson’s and Alzheimer’s diseases). MB has also been proposed as a brain metabolic enhancer because of its action on mitochondrial cytochrome c oxidase. We used in vitro and in vivo approaches to determine how MB affects brain metabolism and hemodynamics. For in vitro, we evaluated the effect of MB on brain mitochondrial function, oxygen consumption, and glucose uptake. For in vivo, we applied neuroimaging and intravenous measurements to determine MB’s effect on glucose uptake, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO2) under normoxic and hypoxic conditions in rats. MB significantly increases mitochondrial complex I–III activity in isolated mitochondria and enhances oxygen consumption and glucose uptake in HT-22 cells. Using positron emission tomography and magnetic resonance imaging (MRI), we observed significant increases in brain glucose uptake, CBF, and CMRO2 under both normoxic and hypoxic conditions. Further, MRI revealed that MB dramatically increased CBF in the hippocampus and in the cingulate, motor, and frontoparietal cortices, areas of the brain affected by Alzheimer’s and Parkinson’s diseases. Our results suggest that MB can enhance brain metabolism and hemodynamics, and multimetric neuroimaging systems offer a noninvasive

Redox potential of the MB related compounds.

<p>Redox potential of the MB related compounds.</p

Different action of MB and 2-chlorophenothiazine on mitochondrial complex IV.

<p>(A) Western blots depict the expression of complex IV subunit I (Cox1) in HT-22 cells treated with MB or 2-chlorophenothiazine at the indicated concentrations for 3 days. MB, at concentrations of 10 and 100 nM, increased Cox1expression. 2-Chlorophenothiazine had no effect on Cox1 expression at 10 nM, 100 nM, and 1 µM. (B). Blue native indicated an increase in complex IV activity at 100 nM MB and a decrease in complex IV activity at 1 µM MB corresponding to the increased expression of Cox1. 2-Chlorophenothiazine had no effect on complex IV activity at all concentrations tested.</p

Schematic illustration depicts the novel neuroprotective mechanism of MB.

<p>MB receives electrons from NADH through mitochondrial complex I and is reduced to leuco-MB, which can donate the electrons to cytochrome c and recycle to its oxidized form, MB. In addition, the reduced leuco-MB can also function as free radical scavenger and neutralize superoxide generated due to the blockage of complex I and III.</p