Inhibition of NADPH oxidase 2 enhances resistance to viral neuroinflammation by facilitating M1-polarization of macrophages at the extraneural tissues.

BACKGROUND: Macrophages play a pivotal role in the regulation of Japanese encephalitis (JE), a severe neuroinflammation in the central nervous system (CNS) following infection with JE virus (JEV). Macrophages are known for their heterogeneity, polarizing into M1 or M2 phenotypes in the context of various immunopathological diseases. A comprehensive understanding of macrophage polarization and its relevance to JE progression holds significant promise for advancing JE control and therapeutic strategies. METHODS: To elucidate the role of NADPH oxidase-derived reactive oxygen species (ROS) in JE progression, we assessed viral load, M1 macrophage accumulation, and cytokine production in WT and NADPH oxidase 2 (NOX2)-deficient mice using murine JE model. Additionally, we employed bone marrow (BM) cell-derived macrophages to delineate ROS-mediated regulation of macrophage polarization by ROS following JEV infection. RESULTS: NOX2-deficient mice exhibited increased resistance to JE progression rather than heightened susceptibility, driven by the regulation of macrophage polarization. These mice displayed reduced viral loads in peripheral lymphoid tissues and the CNS, along with diminished infiltration of inflammatory cells into the CNS, thereby resulting in attenuated neuroinflammation. Additionally, NOX2-deficient mice exhibited enhanced JEV-specific Th1 CD4 + and CD8 + T cell responses and increased accumulation of M1 macrophages producing IL-12p40 and iNOS in peripheral lymphoid and inflamed extraneural tissues. Mechanistic investigations revealed that NOX2-deficient macrophages displayed a more pronounced differentiation into M1 phenotypes in response to JEV infection, thereby leading to the suppression of viral replication. Importantly, the administration of H2O2 generated by NOX2 was shown to inhibit M1 macrophage polarization. Finally, oral administration of the ROS scavenger, butylated hydroxyanisole (BHA), bolstered resistance to JE progression and reduced viral loads in both extraneural tissues and the CNS, along with facilitated accumulation of M1 macrophages. CONCLUSION: In light of our results, it is suggested that ROS generated by NOX2 play a role in undermining the control of JEV replication within peripheral extraneural tissues, primarily by suppressing M1 macrophage polarization. Subsequently, this leads to an augmentation in the viral load invading the CNS, thereby facilitating JE progression. Hence, our findings ultimately underscore the significance of ROS-mediated macrophage polarization in the context of JE progression initiated JEV infection.

Astaxanthin Induces Apoptosis in MCF-7 Cells through a p53-Dependent Pathway.

Astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione; AXT) is a xanthophyll ß-carotenoid found in microalgae, seafood, fungi, complex plants, flamingos, and quail. It is well known that AXT plays a role as a drug with antioxidant and antitumor properties. Furthermore, several studies have reported that the reagent shows anti-inflammatory and neuroprotective effects. Recently, it was found that AXT acts as a peroxisome proliferator-activated receptor γ (PPARγ) modulator. To investigate the effect of AXT on MCF-7 cells (a human breast cancer cell line), the cells were treated with various concentrations of AXT. The treatment induced the decrease in cell number in a dose-dependent manner. Additionally, the Annexin V-positive cells were increased by the AXT treatment. These results indicated that apoptosis was induced in the tumor cells through the treatment of AXT. To elucidate the connection between apoptosis and p53, the levels of p53 and p21 proteins were assessed. Consequently, it was observed that the expression of p53 and p21 increased proportionally to the concentration of the AXT treatment. These findings suggest that the apoptosis of MCF-7 cells induced by AXT operates through a p53-dependent pathway, implying that AXT could potentially have a beneficial role in future breast cancer treatments. Thus, our results will provide a direction for future cancer challenges.

The role of 27-hydroxycholesterol in meta-inflammation.

27-Hydroxycholesterol (27OHChol), a prominent cholesterol metabolite present in the bloodstream and peripheral tissues, is a kind of immune oxysterol that elicits immune response. Recent research indicates the involvement of 27OHChol in metabolic inflammation (meta-inflammation) characterized by chronic responses associated with metabolic irregularities. 27OHChol activates monocytic cells such that they secrete pro-inflammatory cytokines and chemokines, and increase the expression of cell surface molecules such as pattern-recognition receptors that play key roles in immune cell-cell communication and sensing metabolism-associated danger signals. Levels of 27OHChol increase when cholesterol metabolism is disrupted, and the resulting inflammatory responses can contribute to the development and complications of metabolic syndrome, including obesity, insulin resistance, and cardiovascular diseases. Since 27OHChol can induce chronic immune response by activating monocyte-macrophage lineage cells that play a crucial role in meta-inflammation, it is essential to understand the 27OHChol-induced inflammatory responses to unravel the roles and mechanisms of action of this cholesterol metabolite in chronic metabolic disorders.

TLR3/TRIF pathway confers protection against herpes simplex encephalitis through NK cell activation mediated by a loop of type I IFN and IL-15 from epithelial and dendritic cells.

Autosomal recessive (AR) and dominant (AD) deficiencies of TLR3 and TRIF are believed to be crucial genetic causes of herpes simplex encephalitis (HSE), which is a fatal disease causing focal or global cerebral dysfunction following infection with herpes simplex virus type 1 (HSV-1). However, few studies have been conducted on the immunopathological networks of HSE in the context of TLR3 and TRIF defects at the cellular and molecular levels. In this work, we deciphered the crosstalk between type I IFN (IFN-I)-producing epithelial layer and IL-15-producing dendritic cells (DC) to activate NK cells for the protective role of TLR3/TRIF pathway in HSE progression after vaginal HSV-1 infection. TLR3- and TRIF-ablated mice showed enhanced susceptibility to HSE progression, along with high HSV-1 burden in vaginal tract, lymphoid tissues and CNS. The increased HSV-1 burden in TLR3- and TRIF-ablated mice did not correlate with increased infiltration of Ly-6C+ monocytes, but it was closely associated with impaired NK cell activation in vaginal tract. Furthermore, using delicate ex vivo experiments and bone marrow transplantation, TRIF deficiency in tissue-resident cells, such as epithelial cells in vaginal tract, was found to cause impaired NK cell activation by means of low IFN-I production, whereas IFN-I receptor in DC was required for NK cell activation via IL-15 production in response to IFN-I produced from epithelial layer. These results provide new information about IFN-I- and IL-15-mediated crosstalk between epithelial cells and DC at the primary infection site, which suppresses HSE progression in a TLR3- and TRIF-dependent manner.

Side-Chain Immune Oxysterols Induce Neuroinflammation by Activating Microglia.

In individuals with Alzheimer's disease, the brain exhibits elevated levels of IL-1ß and oxygenated cholesterol molecules (oxysterols). This study aimed to investigate the effects of side-chain oxysterols on IL-1ß expression using HMC3 microglial cells and ApoE-deficient mice. Treatment of HMC3 cells with 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol) led to increased IL-1ß expression at the transcript and protein levels. Additionally, these oxysterols upregulated the surface expression of MHC II, a marker of activated microglia. Immunohistochemistry performed on the mice showed increased microglial expression of IL-1ß and MHC II when fed a high-cholesterol diet. However, cholesterol and 24s-hydroxycholesterol did not increase IL-1ß transcript levels or MHC II expression. The extent of IL-1ß increase induced by 25OHChol and 27OHChol was comparable to that caused by oligomeric ß-amyloid, and the IL-1ß expression induced by the oxysterols was not impaired by polymyxin B, which inhibited lipopolysaccharide-induced IL-1ß expression. Both oxysterols enhanced the phosphorylation of Akt, ERK, and Src, and inhibition of these kinase pathways with pharmacological inhibitors suppressed the expression of IL-1ß and MHC II. The pharmacological agents chlorpromazine and cyclosporin A also impaired the oxysterol-induced expression of IL-1ß and upregulation of MHC II. Overall, these findings suggest that dysregulated cholesterol metabolism leading to elevated levels of side-chain oxysterols, such as 25OHChol and 27OHChol, can activate microglia to secrete IL-1ß through a mechanism amenable to pharmacologic intervention. The activation of microglia and subsequent neuroinflammation elicited by the immune oxysterols can contribute to the development of neurodegenerative diseases.

Atheroma-Relevant 7-Oxysterols Differentially Upregulate Cd14 Expression.

The expression of CD14 in monocytic cells is elevated in atherosclerotic lesions where 7-oxyterols are abundant. However, it remains unknown whether atheroma-relevant 7-oxysterols are involved in receptor expression. Therefore, we investigated the effects of 7α-hydroxycholesterol (7αOHChol), 7ß-hydroxycholesterol (7ßOHChol), and 7-ketocholesterol (7K) on CD14 levels in THP-1 cells. The three 7-oxysterols increased CD14 transcript levels at a distinct time point, elevated cellular CD14 protein levels, and promoted the release of soluble CD (sCD14) from THP-1 cells. Our data revealed that CD14 expression was most strongly induced after treatment with 7αOHChol. Moreover, 7αOHChol alone upregulated membrane-bound CD14 levels and enhanced responses to lipopolysaccharides, as determined by CCL2 production and monocytic cell migration. The 7-oxysterols also increased the gelatinolytic activity of MMP-9, and a cell-permeable, reversible MMP-9 inhibitor, MMP-9 inhibitor I, significantly impaired sCD14 release. These results indicate that 7-oxysterols differentially induce CD14 expression in vascular cells and contribute to the monocytic cell expression of CD14 via overlapping, but distinct, mechanisms.

T cell-intrinsic miR-155 is required for Th2 and Th17-biased responses in acute and chronic airway inflammation by targeting several different transcription factors.

Asthmatic airway inflammation is divided into two typical endotypes: Th2-mediated eosinophilic and Th1- or Th17-mediated neutrophilic airway inflammation. The miRNA miR-155 has well-documented roles in the regulation of adaptive T-cell responses and innate immunity. However, no specific cell-intrinsic role has yet been elucidated for miR-155 in T cells in the course of Th2-eosinophilic and Th17-neutrophilic airway inflammation using actual in vivo asthma models. Here, using conditional KO (miR155ΔCD4 cKO) mice that have the specific deficiency of miR-155 in T cells, we found that the specific deficiency of miR-155 in T cells resulted in fully suppressed Th2-type eosinophilic airway inflammation following acute allergen exposure, as well as greatly attenuated the Th17-type neutrophilic airway inflammation induced by repeated allergen exposure. Furthermore, miR-155 in T cells appeared to regulate the expression of several different target genes in the functional activation of CD4+ Th2 and Th17 cells. To be more precise, the deficiency of miR-155 in T cells enhanced the expression of c-Maf, SOCS1, Fosl2 and Jarid2 in the course of CD4+ Th2 cell activation, while C/EBPß was highly enhanced in CD4+ Th17 cell activation in the absence of miR-155 expression. Conclusively, our data revealed that miR-155 could promote Th2 and Th17-mediated airway inflammation via the regulation of several different target genes, depending on the context of asthmatic diseases. Therefore, these results provide valuable insights into actual understanding of specific cell-intrinsic role of miR-155 in eosinophilic and neutrophilic airway inflammation for the development of fine-tune therapeutic strategies.

Type I IFN signaling limits hemorrhage-like disease after infection with Japanese encephalitis virus through modulating a prerequisite infection of CD11b+Ly-6C+ monocytes.

BACKGROUND: The crucial role of type I interferon (IFN-I, IFN-α/ß) is well known to control central nervous system (CNS) neuroinflammation caused by neurotrophic flaviviruses such as Japanese encephalitis virus (JEV) and West Nile virus. However, an in-depth analysis of IFN-I signal-dependent cellular factors that govern CNS-restricted tropism in JEV infection in vivo remains to be elucidated. METHODS: Viral dissemination, tissue tropism, and cytokine production were examined in IFN-I signal-competent and -incompetent mice after JEV inoculation in tissues distal from the CNS such as the footpad. Bone marrow (BM) chimeric models were used for defining hematopoietic and tissue-resident cells in viral dissemination and tissue tropism. RESULTS: The paradoxical and interesting finding was that IFN-I signaling was essentially required for CNS neuroinflammation following JEV inoculation in distal footpad tissue. IFN-I signal-competent mice died after a prolonged neurological illness, but IFN-I signal-incompetent mice all succumbed without neurological signs. Rather, IFN-I signal-incompetent mice developed hemorrhage-like disease as evidenced by thrombocytopenia, functional injury of the liver and kidney, increased vascular leakage, and excessive cytokine production. This hemorrhage-like disease was closely associated with quick viral dissemination and impaired IFN-I innate responses before invasion of JEV into the CNS. Using bone marrow (BM) chimeric models, we found that intrinsic IFN-I signaling in tissue-resident cells in peripheral organs played a major role in inducing the hemorrhage-like disease because IFN-I signal-incompetent recipients of BM cells from IFN-I signal-competent mice showed enhanced viral dissemination, uncontrolled cytokine production, and increased vascular leakage. IFN-I signal-deficient hepatocytes and enterocytes were permissive to JEV replication with impaired induction of antiviral IFN-stimulated genes, and neuron cells derived from both IFN-I signal-competent and -incompetent mice were vulnerable to JEV replication. Finally, circulating CD11b+Ly-6C+ monocytes infiltrated into the distal tissues inoculated by JEV participated in quick viral dissemination to peripheral organs of IFN-I signal-incompetent mice at an early stage. CONCLUSION: An IFN-I signal-dependent model is proposed to demonstrate how CD11b+Ly-6C+ monocytes are involved in restricting the tissue tropism of JEV to the CNS.

27-Hydroxycholesterol induces macrophage gene expression via LXR-dependent and -independent mechanisms.

27-Hydroxycholesterol (27OHChol) exhibits agonistic activity for liver X receptors (LXRs). To determine roles of the LXR agonistic activity in macrophage gene expression, we investigated the effects of LXR inhibition on the 27OHChol-induced genes. Treatment of human THP-1 cells with GSK 2033, a potent cell-active LXR antagonist, results in complete inhibition in the transcription of LXR target genes (such as LXRα and ABCA1) induced by 27OHChol or a synthetic LXR ligand TO 901317. Whereas expression of CCL2 and CCL4 remains unaffected by GSK 2033, TNF-α expression is further induced and 27OHChol-induced CCL3 and CXCL8 genes are suppressed at both the transcriptional and protein translation levels in the presence of GSK 2033. This LXR antagonist downregulates transcript levels and surface expression of CD163 and CD206 and suppresses the transcription of CD14, CD80, and CD86 genes without downregulating their surface levels. GSK 2033 alone had no effect on the basal expression levels of the aforementioned genes. Collectively, these results indicate that LXR inhibition leads to differential regulation of 27-hydroxycholesterolinduced genes in macrophages. We propose that 27OHChol induces gene expression and modulates macrophage functions via LXR-dependent and -independent mechanisms.

CCR5 attenuates neutrophilic airway inflammation exacerbated by infection with rhinovirus.

Human rhinovirus (hRV) is the most common cause of asthma exacerbation characterized by clinical and pathophysiological heterogeneity. Steroid-sensitive, Th2 type-eosinophilic asthma has been somewhat studied, but hRV-induced neutrophilic asthma exacerbation is poorly understood. Here, CCR5 was found to play a role in attenuating neutrophilic airway inflammation in hRV-induced asthma exacerbation using chicken ovalbumin (OVA)-based model. CCR5 deficiency resulted in exacerbated neutrophilic asthmatic responses in airways following hRV infection. CCR5-deficient mice showed enhanced mucus expression and altered expression of tight junction proteins in lung tissues. CCR5-deficient mice were also manifested with influx of CD45+CD11b+Siglec-F+Gr-1+ neutrophils, along with enhanced production of IL-17A, IFN-γ, IL-6, IL-1ß cytokines in inflamed tissues. In contrast, CCR5-deficient mice elicited down-regulation of Th2-related cytokine proteins following hRV infection. More interestingly, the lack of CCR5 altered the equilibrium of CD4+FoxP3+ Tregs and IL-17+CD4+ Th17 in inflamed tissues. CCR5-deficient mice showed increased frequency and absolute number of IL-17-producing CD4+ Th17 cells in lung tissues compared to wild-type mice, whereas the reduced infiltration of CD4+FoxP3+ Treg cells was observed. CCR5 deficiency resulted in the skewed production of Th17 and Th1 cytokines in lymph nodes and lungs upon OVA stimulation. Likewise, CCR5-deficient mice showed enhanced expression of Th17-inducing cytokines (IL-1ß, IL-6, and TNF-α) in lung tissues. These results imply that CCR5 deficiency facilitates Th17 airway inflammation during hRV-induced asthma exacerbation, along with suppressing Th2 responses. Furthermore, our results suggest that CCR5 attenuates hRV-induced neutrophilic airway inflammation through conserving the equilibrium of CD4+Foxp3+ Treg cells and IL-17+CD4+ Th17 cells in hRV-induced asthma exacerbation.

Sildenafil protects neuronal cells from mitochondrial toxicity induced by ß-amyloid peptide via ATP-sensitive K+ channels.

To understand the molecular mechanisms underlying the beneficial effects of sildenafil in animal models of neurological disorders, we investigated the effects of sildenafil on the mitochondrial toxicity induced by ß-amyloid (Aß) peptide. Treatment of HT-22 hippocampal neuronal cells with Aß25∼35 results in increased mitochondrial Ca2+ load, which is subsequently suppressed by sildenafil as well as by diazoxide, a selective opener of the ATP-sensitive K+ channels (KATP). However, the suppressive effects of sildenafil and diazoxide are significantly attenuated by 5-hydroxydecanoic acid (5-HD), a KATP inhibitor. The increased mitochondrial Ca2+ overload is accompanied by decrease in the intracellular ATP concentration, increase in intracellular ROS generation, occurrence of mitochondrial permeability transition, and activation of caspase-9 and cell death. Exposure to sildenafil inhibited the mitochondria-associated changes and cell death induced by Aß. However, the inhibitory effects of sildenafil are abolished or weakened in the presence of 5-HD, suggesting that opening of the mitochondrial KATP is required for sildenafil to exert these effects. Taken together, these results indicate that at the mitochondrial levels, sildenafil plays a protective role towards neuronal cell in an environment rich in Aß, and exerts its effects via the mitochondrial KATP channels-dependent mechanisms.

7-Oxygenated cholesterol molecules differentially affect the expression of zonula occludens-1 in vascular smooth muscle cells and monocyte/macrophage cells.

To investigate the effects of 7-oxygenated cholesterol molecules on the expression of tight junction proteins, we examined the outcomes effects of 7-ketocholesterol (7K), 7α-hydroxycholesterol (7αOHChol) and 7ß-hydroxycholesterol (7ßOHChol) on the expression of the tight-junction protein zonula occludens-1 (ZO-1) using vascular cells. Vascular smooth muscle cells (VSMCs) constitutively express ZO-1, and this expression remained unaffected in the presence of cholesterol. However, the level of ZO-1 protein decreased after exposure to 7K and, to a lesser extent, 7αOHChol and 7ßOHChol. ZO-1 was translocated to the nucleus following treatment with 7K; this translocation was inhibited by z-VAD-fmk, a pan-caspase inhibitor. ZO-1 protein was found to disintegrate in the aorta of ApoE knockout mice fed a high cholesterol diet, whereas it remained intact in the wild-type control. THP-1 monocyte/macrophage cells, which show no expression of ZO-1, were not influenced by treatment with cholesterol, 7K, and 7ßOHChol. However, the treatment of THP-1 cells with 7αOHChol resulted in ZO-1 expression, which largely remained localized on the cytoplasmic membrane. These results indicate the varying effects of 7-oxygenated cholesterol molecules on the expression and localization of ZO-1 depending on cell types, and suggest the contribution of 7-oxygeneted cholesterol molecules to the structural alteration of tight junctions.

PI3K and ERK signaling pathways are involved in differentiation of monocytic cells induced by 27-hydroxycholesterol.

27-Hydroxycholesterol induces differentiation of monocytic cells into mature dendritic cells, mDCs. In the current study we sought to determine roles of the PI3K and the ERK pathways in the 27OHChol-induced differentiation. Up-regulation of mDC-specific markers like CD80, CD83 and CD88 induced by stimulation with 27OHChol was significantly reduced in the presence of LY294002, an inhibitor of PI3K, and U0126, an inhibitor of ERK. Surface expression of MHC class I and II molecules elevated by 27OHChol was decreased to basal levels in the presence of the inhibitors. Treatment with LY294002 or U0126 resulted in recovery of endocytic activity which was reduced by 27OHChol. CD197 expression and cell adherence enhanced by 27OHChol were attenuated in the presence of the inhibitors. Transcription and surface expression of CD molecules involved in atherosclerosis such as CD105, CD137 and CD166 were also significantly decreased by treatment with LY294002 and U0126. These results mean that the PI3K and the ERK signaling pathways are necessary for differentiation of monocytic cells into mDCs and involved in over-expression of atherosclerosis-associated molecules in response to 27OHChol.

27-Hydroxycholesterol up-regulates CD14 and predisposes monocytic cells to superproduction of CCL2 in response to lipopolysaccharide.

We investigated the possibility that a cholesterol-rich milieu can accelerate response to pathogen-associated molecular patterns in order to elucidate mechanisms underlying aggravation of atherosclerosis after bacterial infection. The consumption of a high-cholesterol diet resulted in enhanced the expression of CD14 in arteries of ApoE(-/-) mice. 27-Hydroxycholesterol (27OHChol), the most abundant cholesterol oxide in atherosclerotic lesions, induced the significant expression of CD14 by THP-1 monocytic cells, but not by vascular smooth muscle cells or Jurkat T cells. Additions of lipopolysaccharide (LPS) to 27OHChol-treated THP-1 monocytic cells resulted in superinduction in terms of the gene transcription of CCL2 and the secretion of its gene product. In contrast, cholesterol did not cause increased the expression of CD14 in the aforementioned cells, and the addition of LPS to cholesterol-treated monocytic cells did not result in enhanced the expression of CCL2. The conditioned medium isolated from THP-1 cells exposed to 27OHChol plus LPS further induced the migration of monocytic cells in comparison with conditioned media obtained from THP-1 cells treated with 27OHChol or LPS alone. Treatment with 27OHChol also resulted in the enhanced secretion of MMP-9 and soluble CD14 (sCD14), and the secretion of sCD14 was blocked by a selective MMP-9 inhibitor. The inhibition of the ERK pathway resulted in significantly attenuated the secretion of sCD14 via mechanisms that were distinct from those by PI3K inhibition. We propose that 27OHChol can prime monocytes/macrophages by up-regulation of CD14 such that LPS-mediated inflammatory reaction is accelerated, thereby contributing to aggravated development of atherosclerotic lesions by enhancing recruitment of monocytic cells after infection with Gram-negative bacteria.

CCL2, but not its receptor, is essential to restrict immune privileged central nervous system-invasion of Japanese encephalitis virus via regulating accumulation of CD11b(+) Ly-6C(hi) monocytes.

Japanese encephalitis virus (JEV) is a re-emerging zoonotic flavivirus that poses an increasing threat to global health and welfare due to rapid changes in climate and demography. Although the CCR2-CCL2 axis plays an important role in trafficking CD11b(+) Ly-6C(hi) monocytes to regulate immunopathological diseases, little is known about their role in monocyte trafficking during viral encephalitis caused by JEV infection. Here, we explored the role of CCR2 and its ligand CCL2 in JE caused by JEV infection using CCR2- and CCL2-ablated murine models. Somewhat surprisingly, the ablation of CCR2 and CCL2 resulted in starkly contrasting susceptibility to JE. CCR2 ablation induced enhanced resistance to JE, whereas CCL2 ablation highly increased susceptibility to JE. This contrasting regulation of JE progression by CCR2 and CCL2 was coupled to central nervous system (CNS) infiltration of Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. There was also enhanced expression of CC and CXC chemokines in the CNS of CCL2-ablated mice, which appeared to induce CNS infiltration of these cell populations. However, our data revealed that contrasting regulation of JE in CCR2- and CCL2-ablated mice was unlikely to be mediated by innate natural killer and adaptive T-cell responses. Furthermore, CCL2 produced by haematopoietic stem cell-derived leucocytes played a dominant role in CNS accumulation of Ly-6C(hi) monocytes in infected bone marrow chimeric models, thereby exacerbating JE progression. Collectively, our data indicate that CCL2 plays an essential role in conferring protection against JE caused by JEV infection. In addition, blockage of CCR2, but not CCL2, will aid in the development of strategies for prophylactics and therapeutics of JE.

Diclofenac inhibits 27-hydroxycholesterol-induced inflammation.

27-Hydroxycholesterol (27OHChol) is a cholesterol oxidation product that induces inflammation. In the current study we investigated the effects of diclofenac on inflammatory responses caused by 27OHChol using human monocyte/macrophage (THP-1) cells. Transcription and secretion of CCL2, CCL3, and CCL4 chemokines enhanced by 27OHChol were significantly attenuated by diclofenac in a concentration dependent manner. Migrations of monocytic cells and CCR5-positive Jurkat T cells were reduced proportionally to the concentrations of diclofenac. Superproduction of CCL2 and monocytic cell migration induced by 27OHChol plus LPS were significantly attenuated by diclofenac. Diclofenac also attenuated transcription of MMP-9 and release of its active gene product. These results indicate that diclofenac inhibits 27OHChol-induced inflammatory responses, thereby suppressing inflammation in a milieu rich in cholesterol oxidation products.

Blockage of indoleamine 2,3-dioxygenase regulates Japanese encephalitis via enhancement of type I/II IFN innate and adaptive T-cell responses.

BACKGROUND: Japanese encephalitis (JE), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV). Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function. Although the regulatory role of IDO in viral replication has been postulated, the in vivo role of IDO activity has not been fully addressed in neurotropic virus-caused encephalitis. METHODS: Mice in which IDO activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. Neuroinflammation was evaluated by central nervous system (CNS) infiltration of leukocytes and cytokine expression. IDO expression, viral burden, JEV-specific T-cell, and type I/II interferon (IFN-I/II) innate responses were also analyzed. RESULTS: Elevated expression of IDO activity in myeloid and neuron cells of the lymphoid and CNS tissues was closely associated with clinical signs of JE. Furthermore, inhibition of IDO activity enhanced resistance to JE, reduced the viral burden in lymphoid and CNS tissues, and resulted in early and increased CNS infiltration by Ly-6C(hi) monocytes, NK, CD4(+), and CD8(+) T-cells. JE amelioration in IDO-ablated mice was also associated with enhanced NK and JEV-specific T-cell responses. More interestingly, IDO ablation induced rapid enhancement of type I IFN (IFN-I) innate responses in CD11c(+) dendritic cells (DCs), including conventional and plasmacytoid DCs, following JEV infection. This enhanced IFN-I innate response in IDO-ablated CD11c(+) DCs was coupled with strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7, STAT1), and antiviral ISG genes (Mx1, Mx2, ISG49, ISG54, ISG56). IDO ablation also enhanced the IFN-I innate response in neuron cells, which may delay the spread of virus in the CNS. Finally, we identified that IDO ablation in myeloid cells derived from hematopoietic stem cells (HSCs) dominantly contributed to JE amelioration and that HSC-derived leukocytes played a key role in the enhanced IFN-I innate responses in the IDO-ablated environment. CONCLUSIONS: Inhibition of IDO activity ameliorated JE via enhancement of antiviral IFN-I/II innate and adaptive T-cell responses and increased CNS infiltration of peripheral leukocytes. Therefore, our data provide valuable insight into the use of IDO inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses.

Distinct dictation of Japanese encephalitis virus-induced neuroinflammation and lethality via triggering TLR3 and TLR4 signal pathways.

Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3(-/-) and TLR4(-/-) mice, i.e. TLR3(-/-) mice were highly susceptible to JE, whereas TLR4(-/-) mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b(+)Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4(-/-) mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3(-/-) myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4(+) and CD8(+) T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b(+)Ly-6C(high) monocytes) and CD4(+)Foxp3(+) Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.

7α-Hydroxycholesterol induces inflammation by enhancing production of chemokine (C-C motif) ligand 2.

We investigated pro-inflammatory activity of 7-oxygenated cholesterol derivatives present in atherosclerotic lesions. Treatment of THP-1 monocyte/macrophage with 7α-hydroxycholesterol (7αOHChol) resulted in increased gene transcription of CCL2 and production of its corresponding protein. The conditioned medium isolated from THP-1 cells treated with 7αOHChol enhanced migration of monocytic cells, and migration was inhibited in the presence of CCL2-neutralizing antibody. In contrast, 7ß-hydroxycholesterol (7ßOHChol) or 7-ketocholesterol (7K) did not induce expression of CCL2, and the conditioned medium isolated from THP-1 cells exposed to 7ßOHChol or 7K did not affect migration of monocytic cells. 7αOHChol also enhanced production of MMP-9. Inhibition of MEK or PI3K resulted in significantly attenuated expression of CCL2, along with that of MMP-9, induced by 7αOHChol. We propose that elevated concentration of a certain type of 7-oxygenated cholesterol derivative, like 7αOHChol, leads to inflammation via upregulation of CCL2 and MMP-9 in macrophages in the artery, thereby promoting progression of atherosclerosis, and the ERK and the PI3K pathways are involved in the process.

7-Ketocholesterol induces the reduction of KCNMB1 in atherosclerotic blood vessels.

Hypertension is a high-risk symptom in atherosclerotic patients, and vascular rigidity is one of the main factors leading to hypertension. ß1-Subunit of BKCa channel (KCNMB1; MaxiKß1) has been reported as a modulator of vascular flexibility. To determine the relationship between atherosclerosis and KCNMB1, we studied some atherogenic factors affecting vascular tone. Blood of atherosclerotic patients shows increased concentration of 7-ketocholesterol (7K), which has been studied as a harmful lipid to blood vessels. Our data showed that KCNMB1 was significantly down-regulated in the presence of 7K, in a dose-/time-dependent manner in vascular smooth muscle cells (VSMCs). And, the reduction of KCNMB1 was confirmed in cell images of 7K-stimulated VSMCs and in vessel tissue images of ApoE knock-out mice. To determine whether aryl hydrocarbon receptor (AhR) was involved in the reduction of KCNMB1 by 7K-stimulation, protein level of AhR was analyzed by Western blot. Our data showed that the reduction of KCNMB1 was modulated through the AhR pathway. In conclusion, results of our study suggest that 7K induces the reduction of KCNMB1 through the AhR pathway.