DJ-1 inhibits microglial activation and protects dopaminergic neurons in vitro and in vivo through interacting with microglial p65

Parkinson’s disease (PD), one of the most common neurodegenerative disorders, is characterized by progressive neurodegeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). DJ-1 acts essential roles in neuronal protection and anti-neuroinflammatory response, and its loss of function is tightly associated with a familial recessive form of PD. However, the molecular mechanism of DJ-1 involved in neuroinflammation is largely unclear. Here, we found that wild-type DJ-1, rather than the pathogenic L166P mutant DJ-1, directly binds to the subunit p65 of nuclear factor-κB (NF-κB) in the cytoplasm, and loss of DJ-1 promotes p65 nuclear translocation by facilitating the dissociation between p65 and NF-κB inhibitor α (IκBα). DJ-1 knockout (DJ-1^−/−) mice exhibit more microglial activation compared with wild-type littermate controls, especially in response to lipopolysaccharide (LPS) treatment. In cellular models, knockdown of DJ-1 significantly upregulates the gene expression and increases the release of LPS-treated inflammatory cytokines in primary microglia and BV2 cells. Furthermore, DJ-1 deficiency in microglia significantly enhances the neuronal toxicity in response to LPS stimulus. In addition, pharmacological blockage of NF-κB nuclear translocation by SN-50 prevents microglial activation and alleviates the damage of DA neurons induced by microglial DJ-1 deficiency in vivo and in vitro. Thus, our data illustrate a novel mechanism by which DJ-1 facilitates the interaction between IκBα and p65 by binding to p65 in microglia, and thus repressing microglial activation and exhibiting the protection of DA neurons from neuroinflammation-mediated injury in PD.Subject terms: Cell death in the nervous system, Parkinson''s disease     

Liver Kinase B1 Suppresses Lipopolysaccharide-induced Nuclear Factor κB (NF-κB) Activation in Macrophages

Liver kinase B1 (LKB1), a serine/threonine kinase, is a tumor suppressor and metabolic regulator. Recent data suggest that LKB1 is essential in regulating homeostasis of hematopoietic cells and immune responses. However, its role in macrophages and innate immune system remains unclear. Here we report that macrophage LKB1 inhibits pro-inflammatory signaling in response to LPS. LPS-induced pro-inflammatory cytokines and pro-inflammatory enzymes were monitored in bone marrow-derived macrophages isolated from myeloid cell-specific LKB1 knock out mice and their wild type littermate control mice. LPS induced higher levels of pro-inflammatory cytokines and pro-inflammatory enzymes in bone marrow-derived macrophages from LKB1 KO than those from wild type mice. Consistently, LPS induced higher levels of NF-κB activation in LKB1-deficient macrophages than those in wild type. Further, LPS stimulation significantly increased LKB1 phosphorylation at serine 428, which promoted its binding to IκB kinaseβ (IKKβ), resulting in the inhibition of NF-κB. Finally, LPS injection caused higher levels of cytokine release and more severe tissue injury in the lung tissues of LKB1 KO mice than in those of control mice. We conclude that LKB1 inhibits LPS-induced NF-κB activation in macrophages.     

Local Immunosuppressive Microenvironment Enhances Migration of Melanoma Cells to Lungs in DJ-1 Knockout Mice

DJ-1 is an oncoprotein that promotes survival of cancer cells through anti-apoptosis. However, DJ-1 also plays a role in regulating IL-1β expression, and whether inflammatory microenvironment built by dysregulated DJ-1 affects cancer progression is still unclear. This study thus aimed to compare the metastatic abilities of melanoma cells in wild-type (WT) and DJ-1 knockout (KO) mice, and to check whether inflammatory microenvironment built in DJ-1 KO mice plays a role in migration of cancer cells to lungs. First, B16F10 melanoma cells (at 6×10⁴) were injected into the femoral vein of mice, and formation of lung nodules, levels of lung IL-1β and serum cytokines, and accumulation of myeloid-derived suppressor cells (MDSCs) were compared between WT and DJ-1 KO mice. Second, the cancer-bearing mice were treated with an interleukin-1 beta (IL-1β) neutralizing antibody to see whether IL-1β is involved in the cancer migration. Finally, cultured RAW 264.7 macrophage and B16F10 melanoma cells were respectively treated with DJ-1 shRNA and recombinant IL-1β to explore underlying molecular mechanisms. Our results showed that IL-1β enhanced survival and colony formation of cultured melanoma cells, and that IL-1β levels were elevated both in DJ-1 KO mice and in cultured macrophage cells with DJ-1 knockdown. The elevated IL-1β correlated with higher accumulation of immunosuppressive MDSCs and formation of melanoma module in the lung of DJ-1 KO mice, and both can be decreased by treating mice with IL-1β neutralizing antibodies. Taken together, these results indicate that immunosuppressive tissue microenvironment built in DJ-1 KO mice can enhance lung migration of cancer, and IL-1β plays an important role in promoting the cancer migration.     

Progressive Axonal Degeneration of Nigrostriatal Dopaminergic Neurons in Calcium-Independent Phospholipase A2β Knockout Mice

Calcium-independent phospholipase A₂β (iPLA₂β, PLA2G6) is essential for the remodeling of membrane glycerophospholipids. Mutations in this gene are responsible for autosomal recessive, young onset, L-dopa-responsive parkinsonism (PARK14), suggesting a neurodegenerative condition in the nigrostriatal dopaminergic system in patients with PLA2G6 mutations. We previously observed slowly progressive motor deficits in iPLA₂β-knockout (KO) mice. To clarify whether a deficiency of iPLA₂β leads to the degeneration of nigrostriatal dopaminergic neurons, we analyzed the striatum of iPLA₂β-KO mice. At all clinical stages, nerve terminals in the striatum were immunopositive for tyrosine hydroxylase (TH) and dopamine transporter (DAT) in wild-type (WT) control mice. In iPLA₂β-KO mice, focal loss of nerve terminals positive for TH and DAT was found from 56 weeks (early clinical stage), although iPLA₂β-KO mice at 56 weeks showed no significant decrease in the number of dopaminergic neurons in the substantia nigra compared with age-matched WT mice, as reported previously. At 100 weeks (late clinical stage), greater decreases in DAT immunoreactivity were observed in the striatum of iPLA₂β-KO mice. Moreover, strongly TH-positive structures, presumed to be deformed axons, were observed in the neuropils of the striatum of iPLA₂β-KO mice starting at 15 weeks (preclinical stage) and increased with age. These results suggest that the degeneration of dopaminergic neurons occurs mainly in the distal region of axons in iPLA₂β-KO mice.     

Visualizing Production of Beta Interferon by Astrocytes and Microglia in Brain of La Crosse Virus-Infected Mice

Beta interferon (IFN-β) is a major component of innate immunity in mammals, but information on the in vivo source of this cytokine after pathogen infection is still scarce. To identify the cell types responsible for IFN-β production during viral encephalitis, we used reporter mice that express firefly luciferase under the control of the IFN-β promoter and stained organ sections with luciferase-specific antibodies. Numerous luciferase-positive cells were detected in regions of La Crosse virus (LACV)-infected mouse brains that contained many infected cells. Double-staining experiments with cell-type-specific markers revealed that similar numbers of astrocytes and microglia of infected brains were luciferase positive, whereas virus-infected neurons rarely contained detectable levels of luciferase. Interestingly, if a mutant LACV unable of synthesizing the IFN-antagonistic factor NSs was used for challenge, the vast majority of the IFN-β-producing cells in infected brains were astrocytes rather than microglia. Similar conclusions were reached in a second series of experiments in which conditional reporter mice expressing the luciferase reporter gene solely in defined cell types were infected with wild-type or mutant LACV. Collectively, our data suggest that glial cells rather than infected neurons represent the major source of IFN-β in LACV-infected mouse brains. They further indicate that IFN-β synthesis in astrocytes and microglia is differentially affected by the viral IFN antagonist, presumably due to differences in LACV susceptibility of these two cell types.     

Inhibition of UII/UTR System Relieves Acute Inflammation of Liver through Preventing Activation of NF-κB Pathway in ALF Mice

Urotensin II (UII) is implicated in immune inflammatory diseases through its specific high-affinity UT receptor (UTR). Enhanced expression of UII/UTR was recently demonstrated in the liver with acute liver failure (ALF). Here, we analysed the relationship between UII/UTR expression and ALF in lipopolysaccharide (LPS)/D-galactosamine (GalN)-challenged mice. Thereafter, we investigated the effects produced by the inhibition of UII/UTR system using urantide, a special antagonist of UTR, and the potential molecular mechanisms involved in ALF. Urantide was administered to mice treated with LPS/GalN. Expression of UII/UTR, releases of proinflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interferon-γ (IFN-γ), and activation of nuclear factor κB (NF-κB) signaling pathway were assessed in the lethal ALF with or without urantide pretreatment. We found that LPS/GalN-challenged mice showed high mortality and marked hepatic inflammatory infiltration and cell apoptosis as well as a significant increase of UII/UTR expression. Urantide pretreatment protected against the injury in liver following downregulation of UII/UTR expression. A close relationship between the acutely flamed hepatic injury and UII/UTR expression was observed. In addition, urantide prevented the increases of proinflammatory cytokines such as TNF-α, IL-1β and IFN-γ, and activation of NF-κB signaling pathway induced by LPS/GalN in mice. Thus, we conclude that UII/UTR system plays a role in LPS/GalN-induced ALF. Urantide has a protective effect on the acutely inflamed injury of liver in part through preventing releases of proinflammatory cytokines and activation of NF-κB pathway.     

Interferon Gamma Suppresses Collagen-Induced Arthritis by Regulation of Th17 through the Induction of Indoleamine-2,3-Deoxygenase

C57BL/6 mice are known to be resistant to the development of collagen-induced arthritis (CIA). However, they show a severe arthritic phenotype when the Ifng gene is deleted. Although it has been proposed that IFN-γ suppresses inflammation in CIA via suppressing Th17 which is involved in the pathogenesis of CIA, the exact molecular mechanism of the Th17 regulation by IFN-γ is poorly understood. This study was conducted to 1) clarify that arthritogenic condition of IFN-γ knockout (KO) mice is dependent on the disinhibition of Th17 and 2) demonstrate that IFN-γ-induced indoleamine2,3dioxgenase (IDO) is engaged in the regulation of Th17. The results showed that the IFN-γ KO mice displayed increased levels of IL-17 producing T cells and the exacerbation of arthritis. Also, production of IL-17 by the splenocytes of the IFN-γ KO mice was increased when cultured with type II collagen. When Il17 was deleted from the IFN-γ KO mice, only mild arthritis developed without any progression of the arthritis score. The proportion of CD44^highCD62L^low memory-like T cells were elevated in the spleen, draining lymph node and mesenteric lymph node of IFN-γ KO CIA mice. Meanwhile, CD44^lowCD62L^high naïve T cells were increased in IFN-γ and IL-17 double KO CIA mice. When Th17 polarized CD4+ T cells of IFN-γ KO mice were co-cultured with their own antigen presenting cells (APCs), a greater increase in IL-17 production was observed than in co-culture of the cells from wild type mice. In contrast, when APCs from IFN-γ KO mice were pretreated with IFN-γ, there was a significant reduction in IL-17 in the co-culture system. Of note, pretreatment of 1-methyl-DL- tryptophan, a specific inhibitor of IDO, abolished the inhibitory effects of IFN-γ. Given that IFN-γ is a potent inducer of IDO in APCs, these results suggest that IDO is involved in the regulation of IL-17 by IFN-γ.     

Chronic Alcohol-Induced microRNA-155 Contributes to Neuroinflammation in a TLR4-Dependent Manner in Mice

Introduction

Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation.

Aim

To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation.

Methods

Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation.

Results

Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155.

Conclusion

Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.     

Modelling Neuroinflammation In Vitro: A Tool to Test the Potential Neuroprotective Effect of Anti-Inflammatory Agents

Neuron-microglia co-cultures treated with pro-inflammatory agents are a useful tool to study neuroinflammation in vitro, where to test the potential neuroprotective effect of anti-inflammatory compounds. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. We compared the use of neuron-primary microglia and neuron-BV2 cells (a microglial cell line) co-cultures, using different neuron:microglia ratios, treatments and time post-treatment to induce glial activation and derived neurotoxicity. We show that each model requires different experimental conditions, but that both neuron-BV2 and neuron-primary microglia LPS/IFN-γ-treated co-cultures are good to study the potential neuroprotective effect of anti-inflammatory agents. The contribution of different pro-inflammatory parameters in the neurotoxicity induced by reactive microglial cells was determined. IL-10 pre-treatment completely inhibited LPS/IFN-γ-induced TNF-α and IL-6 release, and COX-2 expression both in BV2 and primary microglial cultures, but not NO production and iNOS expression. However, LPS/IFN-γ induced neurotoxicity was not inhibited in IL-10 pre-treated co-cultures. The inhibition of NO production using the specific iNOS inhibitor 1400 W totally abolished the neurotoxic effect of LPS/IFN-γ, suggesting a major role for NO in the neurotoxic effect of activated microglia. Consequently, among the anti-inflammatory agents, special attention should be paid to compounds that inhibit NO production.     

Yohimbine Enhances Protection of Berberine against LPS-Induced Mouse Lethality through Multiple Mechanisms

Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.     

Activation of BV2 microglia by lipopolysaccharide triggers an inflammatory reaction in PC12 cell apoptosis through a toll-like receptor 4-dependent pathway

Microglia play an important role in neuronal protection and damage. However, the molecular and cellular relationship between microglia and neurons is unclear. We carried out a prospective study to detect that activation of BV2 microglia induced PC12 cell apoptosis in vitro through the TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. BV2 microglia were treated with different concentrations of LPS for 24 h. Western blot was utilized to detect the expression of TLR4 and the downstream signaling pathway. The level of inflammatory mediator was quantified using a specific ELISA kit. The supernatant of 10 μg/ml LPS-treated BV2 cells was used as conditioned medium (CM). PC12 cells were co-culture with CM for 24 h. Cell viability was determined by MTT assay and cell apoptosis was tested by flow cytometry. BV2 microglia were treated with 10, 20, or 30 μg/ml LPS for 24 h. The expression of TLR4, MyD88, and NF-κB significantly increased. When PC12 cells were co-cultured with CM for 24 h, cell viability decreased. CM up-regulated the Bax level and down-regulated the Bcl-2 protein level in PC12 cells. PC12 cells pretreated with interleukin-1 receptor antagonist (IL-1RA) for 30 min, significantly alleviated CM-induced PC12 cell apoptosis. These results suggest that BV2 microglia activated by LPS triggered TLR4/MyD88/NF-κB signaling pathway that induced the release of IL-1β and could participate in the PC12 cells injury.     

Role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice

Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-α/β and IFN-γ receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B- or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-α/β receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-γ receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.     

Tim-4 Inhibits NO Generation by Murine Macrophages

Objective

T cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) receives much attention as a potentially negative regulator of immune responses. However, its modulation on macrophages has not been fully elucidated so far. This study aimed to identify the role of Tim-4 in nitric oxide (NO) modulation.

Methods

Macrophages were stimulated with 100 ng/ml LPS or 100 U/ml IFN-γ. RT-PCR was performed to detect TIM-4 mRNA expression. Tim-4 blocking antibody and NF-κB inhibitory ligand were involved in the study. NO levels were assayed by Griess reaction. Phosphorylation of NF-κB, Jak2 or Stat1 was verified by western blot.

Results

Tim-4 was up-regulated in murine macrophages after interferon-gamma (IFN-γ) stimulation. Tim-4 over-expression decreased NO production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS) or IFN-γ-stimulated macrophages. Consistently, Tim-4 blockade promoted LPS or IFN-γ-induced NO secretion and iNOS expression. Tim-4 over-expression decreased LPS-induced nuclear factor kappa B (NF-κB) p65 phosphorylation in macrophages, which was abrogated by NF-κB inhibitory ligand. On the contrary, Tim-4 blocking increased LPS-induced NF-κB signaling, which was also abrogated by NF-κB inhibition. In addition, Tim-4 blockade promoted Jak2 and Stat1 phosphorylation in IFN-γ stimulated macrophages.

Conclusion

These results indicate that Tim-4 is involved in negative regulation of NO production in macrophages, suggesting the critical role of Tim-4 in immune related diseases.     

Activation of CNTF/CNTFRα Signaling Pathway by hRheb(S16H) Transduction of Dopaminergic Neurons In Vivo

Ciliary neurotrophic factor (CNTF) is one of representative neurotrophic factors for the survival of dopaminergic neurons. Its effects are primarily mediated via CNTF receptor α (CNTFRα). It is still unclear whether the levels of CNTFRα change in the substantia nigra of Parkinson’s disease (PD) patients, but CNTF expression shows the remarkable decrease in dopaminergic neurons in the substantia nigra pars compacta (SNpc), suggesting that the support of CNTF/CNTFRα signaling pathway may be a useful neuroprotective strategy for the nigrostriatal dopaminergic projection in the adult brain. Here, we report that transduction of rat SNpc dopaminergic neurons by adeno-associated virus with a gene encoding human ras homolog enriched in brain (hRheb), with an S16H mutation [hRheb(S16H)], significantly upregulated the levels of both CNTF and CNTFRα in dopaminergic neurons. Moreover, the hRheb(S16H)-activated CNTF/CNTFRα signaling pathway was protective against 1-methyl-4-phenylpyridinium-induced neurotoxicity in the nigrostriatal dopaminergic projections. These results suggest that activation of CNTF/CNTFRα signaling pathway by specific gene delivery such as hRheb(S16H) may have therapeutic potential in the treatment of PD.     

Anti-Inflammatory Effects of Progesterone in Lipopolysaccharide-Stimulated BV-2 Microglia

Female sex is associated with improved outcome in experimental brain injury models, such as traumatic brain injury, ischemic stroke, and intracerebral hemorrhage. This implies female gonadal steroids may be neuroprotective. A mechanism for this may involve modulation of post-injury neuroinflammation. As the resident immunomodulatory cells in central nervous system, microglia are activated during acute brain injury and produce inflammatory mediators which contribute to secondary injury including proinflammatory cytokines, and nitric oxide (NO) and prostaglandin E₂ (PGE₂), mediated by inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. We hypothesized that female gonadal steroids reduce microglia mediated neuroinflammation. In this study, the progesterone’s effects on tumor necrosis factor alpha (TNF-α), iNOS, and COX-2 expression were investigated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Further, investigation included nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways. LPS (30 ng/ml) upregulated TNF-α, iNOS, and COX-2 protein expression in BV-2 cells. Progesterone pretreatment attenuated LPS-stimulated TNF-α, iNOS, and COX-2 expression in a dose-dependent fashion. Progesterone suppressed LPS-induced NF-κB activation by decreasing inhibitory κBα and NF-κB p65 phosphorylation and p65 nuclear translocation. Progesterone decreased LPS-mediated phosphorylation of p38, c-Jun N-terminal kinase and extracellular regulated kinase MAPKs. These progesterone effects were inhibited by its antagonist mifepristone. In conclusion, progesterone exhibits pleiotropic anti-inflammatory effects in LPS-stimulated BV-2 microglia by down-regulating proinflammatory mediators corresponding to suppression of NF-κB and MAPK activation. This suggests progesterone may be used as a potential neurotherapeutic to treat inflammatory components of acute brain injury.     

Targeted Deletion of Growth Hormone (GH) Receptor in Macrophage Reveals Novel Osteopontin-mediated Effects of GH on Glucose Homeostasis and Insulin Sensitivity in Diet-induced Obesity

We investigated GH action on macrophage (MΦ) by creating a MΦ-specific GH receptor-null mouse model (MacGHR KO). On a normal diet (10% fat), MacGHR KO and littermate controls exhibited similar growth profiles and glucose excursions on intraperitoneal glucose (ipGTT) and insulin tolerance (ITT) tests. However, when challenged with high fat diet (HFD, 45% fat) for 18 weeks, MacGHR KO mice exhibited impaired ipGTT and ITT compared with controls. In MacGHR KO, adipose-tissue (AT) MΦ abundance was increased with skewing toward M1 polarization. Expression of pro-inflammatory cytokines (IL1β, TNF-α, IL6, and osteopontin (OPN)) were increased in MacGHR KO AT stromal vascular fraction (SVF). In MacGHR KO AT, crown-like-structures were increased with decreased insulin-dependent Akt phosphorylation. The abundance of phosphorylated NF-κB and of OPN was increased in SVF and bone-marrow-derived MΦ in MacGHR KO. GH, acting via an NF-κB site in the distal OPN promoter, inhibited the OPN promoter. Thus in diet-induced obesity (DIO), lack of GH action on the MΦ exerts an unexpected deleterious effect on glucose homeostasis by accentuating AT inflammation and NF-κB-dependent activation of OPN expression. These novel results in mice support the possibility that administration of GH could have salutary effects on DIO-associated chronic inflammation and insulin resistance in humans.     

Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia

The coronavirus mouse hepatitis virus (MHV) induces a minimal type I interferon (IFN) response in several cell types in vitro despite the fact that the type I IFN response is important in protecting the mouse from infection in vivo. When infected with MHV, mice deficient in IFN-associated receptor expression (IFNAR^−/−) became moribund by 48 h postinfection. MHV also replicated to higher titers and exhibited a more broad tissue tropism in these mice, which lack a type I IFN response. Interestingly, MHV induced IFN-β in the brains and livers, two main targets of MHV replication, of infected wild-type mice. MHV infection of primary cell cultures indicates that hepatocytes are not responsible for the IFN-β production in the liver during MHV infection. Furthermore, macrophages and microglia, but not neurons or astrocytes, are responsible for IFN-β production in the brain. To determine the pathway by which MHV is recognized in macrophages, IFN-β mRNA expression was quantified following MHV infection of a panel of primary bone marrow-derived macrophages generated from mice lacking different pattern recognition receptors (PRRs). Interestingly, MDA5, a PRR thought to recognize primarily picornaviruses, was required for recognition of MHV. Thus, MHV induces type I IFN in macrophages and microglia in the brains of infected animals and is recognized by an MDA5-dependent pathway in macrophages. These findings suggest that secretion of IFN-β by macrophages and microglia plays a role in protecting the host from MHV infection of the central nervous system.