CD40L protects against mouse hepatitis virus-induced neuroinflammatory demyelination

Neurotropic mouse hepatitis virus (MHV-A59/RSA59) infection in mice induces acute neuroinflammation due to direct neural cell dystrophy, which proceeds with demyelination with or without axonal loss, the pathological hallmarks of human neurological disease, Multiple sclerosis (MS). Recent studies in the RSA59-induced neuroinflammation model of MS showed a protective role of CNS-infiltrating CD4⁺ T cells compared to their pathogenic role in the autoimmune model. The current study further investigated the molecular nexus between CD4⁺ T cell-expressed CD40Ligand and microglia/macrophage-expressed CD40 using CD40L^-/- mice. Results demonstrate CD40L expression in the CNS is modulated upon RSA59 infection. We show evidence that CD40L^-/- mice are more susceptible to RSA59 induced disease due to reduced microglia/macrophage activation and significantly dampened effector CD4⁺ T recruitment to the CNS on day 10 p.i. Additionally, CD40L^-/- mice exhibited severe demyelination mediated by phagocytic microglia/macrophages, axonal loss, and persistent poliomyelitis during chronic infection, indicating CD40-CD40L as host-protective against RSA59-induced demyelination. This suggests a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to immunosuppression which holds only for autoimmune mechanisms of inflammatory demyelination.     

NKG2D receptor signaling enhances cytolytic activity by virus-specific CD8+ T cells: evidence for a protective role in virus-induced encephalitis

Inoculation with the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the central nervous system (CNS) of mice results in an acute encephalitis associated with an immune-mediated demyelinating disease. During acute disease, infiltrating CD8⁺ T cells secrete gamma interferon (IFN-γ) that controls replication in oligodendrocytes, while infected astrocytes and microglia are susceptible to perforin-mediated lysis. The present study was undertaken to reveal the functional contributions of the activating NKG2D receptor in host defense and disease following JHMV infection. NKG2D ligands RAE-1, MULT1, and H60 were expressed within the CNS following JHMV infection. The immunophenotyping of infiltrating cells revealed that NKG2D was expressed on ~90% of infiltrating CD8⁺ T cells during acute and chronic disease. Blocking NKG2D following JHMV infection resulted in increased mortality that correlated with increased viral titers within the CNS. Anti-NKG2D treatment did not alter T-cell infiltration into the CNS or the generation of virus-specific CD8⁺ T cells, and the expression of IFN-γ was not affected. However, cytotoxic T-lymphocyte (CTL) activity was dependent on NKG2D expression, because anti-NKG2D treatment resulted in a dramatic reduction in lytic activity by virus-specific CD8⁺ T cells. Blocking NKG2D during chronic disease did not affect either T-cell or macrophage infiltration or the severity of demyelination, indicating that NKG2D does not contribute to virus-induced demyelination. These findings demonstrate a functional role for NKG2D in host defense during acute viral encephalitis by selectively enhancing CTL activity by infiltrating virus-specific CD8⁺ T cells.     

RNase L Mediated Protection from Virus Induced Demyelination

IFN-α/β plays a critical role in limiting viral spread, restricting viral tropism and protecting mice from neurotropic coronavirus infection. However, the IFN-α/β dependent mechanisms underlying innate anti-viral functions within the CNS are poorly understood. The role of RNase L in viral encephalomyelitis was explored based on its functions in inhibiting translation, inducing apoptosis, and propagating the IFN-α/β pathway through RNA degradation intermediates. Infection of RNase L deficient (RL^−/−) mice with a sub-lethal, demyelinating mouse hepatitis virus variant revealed that the majority of mice succumbed to infection by day 12 p.i. However, RNase L deficiency did not affect overall control of infectious virus, or diminish IFN-α/β expression in the CNS. Furthermore, increased morbidity and mortality could not be attributed to altered proinflammatory signals or composition of cells infiltrating the CNS. The unique phenotype of infected RL^−/− mice was rather manifested in earlier onset and increased severity of demyelination and axonal damage in brain stem and spinal cord without evidence for enhanced neuronal infection. Increased tissue damage coincided with sustained brain stem infection, foci of microglia infection in grey matter, and increased apoptotic cells. These data demonstrate a novel protective role for RNase L in viral induced CNS encephalomyelitis, which is not reflected in overall viral control or propagation of IFN-α/β mediated signals. Protective function is rather associated with cell type specific and regional restriction of viral replication in grey matter and ameliorated neurodegeneration and demyelination.     

IFN-Gamma Inhibits JC Virus Replication in Glial Cells by Suppressing T-Antigen Expression

Objective

Patients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), an often lethal disease of the brain characterized by lytic infection of oligodendrocytes in the central nervous system (CNS) with JC virus (JCV). The immune system plays an important regulatory role in controlling JCV reactivation from latent sites by limiting viral gene expression and replication. However, little is known regarding the molecular mechanisms responsible for this regulation.

Methods and Results

Here, we investigated the impact of soluble immune mediators secreted by activated PBMCs on viral replication and gene expression by cell culture models and molecular virology techniques. Our data revealed that viral gene expression and viral replication were suppressed by soluble immune mediators. Further studies demonstrated that soluble immune mediators secreted by activated PBMCs inhibit viral replication induced by T-antigen, the major viral regulatory protein, by suppressing its expression in glial cells. This unexpected suppression of T-antigen was mainly associated with the suppression of translational initiation. Cytokine/chemokine array studies using conditioned media from activated PBMCs revealed several candidate cytokines with possible roles in this regulation. Among them, only IFN-γ showed a robust inhibition of T-antigen expression. While potential roles for IFN-β, and to a lesser extent IFN-α have been described for JCV, IFN-γ has not been previously implicated. Further analysis of IFN-γ signaling pathway revealed a novel role of Jak1 signaling in control of viral T-antigen expression. Furthermore, IFN-γ suppressed JCV replication and viral propagation in primary human fetal glial cells, and showed a strong anti-JCV activity.

Conclusions

Our results suggest a novel role for IFN-γ in the regulation of JCV gene expression via downregulation of the major viral regulatory protein, T-antigen, and provide a new avenue of research to understand molecular mechanisms for downregulation of viral reactivation that may lead to development of novel strategies for the treatment of PML.     

Memory CD4+ T-cell-mediated protection from lethal coronavirus encephalomyelitis

The antiviral role of CD4⁺ T cells in virus-induced pathologies of the central nervous system (CNS) has not been explored extensively. Control of neurotropic mouse hepatitis virus (JHMV) requires the collaboration of CD4⁺ and CD8⁺ T cells, with CD8⁺ T cells providing direct perforin and gamma interferon (IFN-γ)-mediated antiviral activity. To distinguish bystander from direct antiviral contributions of CD4⁺ T cells in virus clearance and pathology, memory CD4⁺ T cells purified from wild type (wt), perforin-deficient (PKO), and IFN-γ-deficient (GKO) immune donors were transferred to immunodeficient SCID mice prior to CNS challenge. All three donor CD4⁺ T-cell populations controlled CNS virus replication at 8 days postinfection, indicating IFN-γ- and perforin-independent antiviral function. Recipients of GKO CD4⁺ T cells succumbed more rapidly to fatal disease than untreated control infected mice. In contrast, wt and PKO donor CD4⁺ T cells cleared infectious virus to undetectable levels and protected from fatal disease. Recipients of all CD4⁺ T-cell populations exhibited demyelination. However, it was more severe in wt CD4⁺ T-cell recipients. These data support a role of CD4⁺ T cells in virus clearance and demyelination. Despite substantial IFN-γ-independent antiviral activity, IFN-γ was crucial in providing protection from death. IFN-γ reduced neutrophil accumulation and directed macrophages to white matter but did not ameliorate myelin loss.     

Expression of OX40 ligand in microglia activated by IFN-gamma sustains a protective CD4+ T-cell response in vitro

T-cell-dependent immunity in the central nervous system (CNS) is beneficial for neuroprotection, neurogenesis and even behavior. As a highly specialized site, the CNS is speculated to possess the means to maintain T-cell immune responses through its own resident cells. Therefore, we investigated whether microglia, the most potent antigen-presenting cells residing in the CNS, could sustain T-cell responses in vitro. We showed that interferon-γ (IFN-γ)-activated microglia (MG_IFN-γ) inducibly expressed an important immune co-stimulatory molecule, OX40 ligand (OX40L). Co-culture of activated CD4⁺ T cells with MG_IFN-γ significantly increased T-cell proliferation and decreased apoptosis, and these effects were markedly inhibited by addition of a neutralizing anti-OX40L monoclonal antibody. In addition, ligation of OX40L in MG_IFN-γ enhanced their production of insulin-like growth factor I (IGF-I). These results suggest that the expression of OX40L in microglia provides a molecular basis for the maintenance of T-cell survival, expansion of T cells and increased secretion of remedial growth factor from MG_IFN-γ, which may contribute to the protective effect in the CNS.     

Regulation of CD47 expression by interferon-gamma in cancer cells

The anti-phagocytosis signal, CD47, prevents phagocytosis when it interacts with signal-regulatory protein alpha (SIRPα) on macrophages. Given the vital role of CD47 in immune response, further investigation on the regulation of CD47 in tumor microenvironment is needed. Herein, we identified that interferon-gamma (IFN-γ), one of the most important cytokines in the immune and inflammatory response, up-regulated CD47 expression in cancer cells and this effect could be inhibited by the JAK1/2 inhibitor ruxolitinib, as well as siRNA-mediated silencing of JAK1, STAT1, and IRF1. The IFN-γ-induced surface expression of CD47 contributed to a stronger binding affinity to SIRPα and a decrease in phagocytosis of cancer cells by macrophages. Knockdown of JAK1, STAT1, or IRF1 by siRNA reversed the decreased phagocytosis caused by IFN-γ. Besides, analysis from TCGA revealed that IFNG had a positive correlation with CD47 in various types of cancer, which was supported by the increased surface CD47 expression after IFN-γ treatment in different types of cancer cells. The discovery of IFN-γ-induced up-regulation of CD47 in cancer cells unveils another feedback inhibitory mechanism of IFN-γ, thus providing insights into cancer immunotherapy targeting CD47.     

Cudrania cochinchinensis attenuates amyloid β protein-mediated microglial activation and promotes glia-related clearance of amyloid β protein

Background

Microglial inflammation may significantly contribute to the pathology of Alzheimer’s disease. To examine the potential of Cudrania cochinchinensis to ameliorate amyloid β protein (Aβ)-induced microglia activation, BV-2 microglial cell line, and the ramified microglia in the primary glial mixed cultured were employed.

Results

Lipopolysaccharide (LPS), Interferon-γ (IFN-γ), fibrillary Aβ (fAβ), or oligomeric Aβ (oAβ) were used to activate microglia. LPS and IFN-γ, but not Aβs, activated BV-2 cells to produce nitric oxide through an increase in inducible nitric oxide synthase (iNOS) expression without significant effects on cell viability of microglia. fAβ, but not oAβ, enhanced the IFN-γ-stimulated nitric oxide production and iNOS expression.The ethanol/water extracts of Cudrania cochinchinensis (CC-EW) and the purified isolated components (i.e. CCA to CCF) effectively reduced the nitric oxide production and iNOS expression stimulated by IFN-γ combined with fAβ. On the other hand, oAβ effectively activated the ramified microglia in mixed glial culture by observing the morphological alteration of the microglia from ramified to amoeboid. CC-EW and CCB effectively prohibit the Aβ-mediated morphological change of microglia. Furthermore, CC-EW and CCB effectively decreased Aβ deposition and remained Aβ in the conditioned medium suggesting the effect of CC-EW and CCB on promoting Aβ clearance. Results are expressed as mean ± S.D. and were analyzed by ANOVA with post-hoc multiple comparisons with a Bonferroni test.

Conclusions

The components of Cudrania cochinchinensis including CC-EW and CCB are potential for novel therapeutic intervention for Alzheimer’s disease.     

Ifit2 Deficiency Results in Uncontrolled Neurotropic Coronavirus Replication and Enhanced Encephalitis via Impaired Alpha/Beta Interferon Induction in Macrophages

Type I interferons (IFN-α/β) limit viral dissemination prior to the emergence of adaptive immune responses through the concerted action of interferon-stimulated genes (ISGs). Although IFN-α/β induction by coronaviruses is modest, it effectively limits viral spread within the central nervous system (CNS) and protects against mortality. The protective roles of specific ISGs against the mouse hepatitis virus (MHV) members of the coronaviruses are largely unknown. This study demonstrates a protective role of the ISG Ifit2 in encephalitis induced by the dual hepato- and neurotropic MHV-A59. Contrasting the mild encephalitis and 100% survival of MHV-A59-infected wild-type (wt) mice, nearly 60% of infected Ifit2^−/− mice exhibited severe encephalitis and succumbed between 6 and 8 days postinfection. Increased clinical disease in Ifit2^−/− mice coincided with higher viral loads and enhanced viral spread throughout the CNS parenchyma. Ifit2^−/− mice also expressed significantly reduced IFN-α/β and downstream ISG mRNAs Ifit1, Isg15, and Pkr, while expression of proinflammatory cytokines and chemokines was only modestly affected in the CNS. Impaired IFN-α/β induction in the absence of Ifit2 was confirmed by ex vivo mRNA analysis of microglia and macrophages, the prominent cell types producing IFN-α/β following MHV CNS infection. Furthermore, both IFN-α/β mRNA and protein production were significantly reduced in MHV-infected Ifit2^−/− relative to wt bone marrow-derived macrophages. Collectively, the data implicate Ifit2 as a positive regulator of IFN-α/β expression, rather than direct antiviral mediator, during MHV-induced encephalitis.     

Activated CD8+ T Lymphocytes Inhibit Neural Stem/Progenitor Cell Proliferation: Role of Interferon-Gamma

The ability of neural stem/progenitor cells (NSCs) to self-renew, migrate to damaged sites, and differentiate into neurons has renewed interest in using them in therapies for neurodegenerative disorders. Neurological diseases, including viral infections of the brain, are often accompanied by chronic inflammation, whose impact on NSC function remains unexplored. We have previously shown that chronic neuroinflammation, a hallmark of experimental herpes simplex encephalitis (HSE) in mice, is dominated by brain-infiltrating activated CD8 T-cells. In the present study, activated CD8 lymphocytes were found to suppress NSC proliferation profoundly. Luciferase positive (luc⁺) NSCs co-cultured with activated, MHC-matched, CD8⁺ lymphocytes (luc⁻) showed two- to five-fold lower luminescence than co-cultures with un-stimulated lymphocytes. On the other hand, similarly activated CD4⁺ lymphocytes did not suppress NSC growth. This differential lymphocyte effect on proliferation was confirmed by decreased BrdU uptake by NSC cultured with activated CD8 T-cells. Interestingly, neutralizing antibodies to interferon-gamma (IFN-γ) reversed the impact of CD8 lymphocytes on NSCs. Antibodies specific to the IFN-γ receptor-1 subunit complex abrogated the inhibitory effects of both CD8 lymphocytes and IFN-γ, indicating that the inhibitory effect of these cells was mediated by IFN-γ in a receptor-specific manner. In addition, activated CD8 lymphocytes decreased levels of nestin and Sox2 expression in NSCs while increasing GFAP expression, suggesting possible induction of an altered differentiation state. Furthermore, NSCs obtained from IFN-γ receptor-1 knock-out embryos were refractory to the inhibitory effects of activated CD8⁺ T lymphocytes on cell proliferation and Sox2 expression. Taken together, the studies presented here demonstrate a role for activated CD8 T-cells in regulating NSC function mediated through the production of IFN-γ. This cytokine may influence neuro-restorative processes and ultimately contribute to the long-term sequelae commonly seen following herpes encephalitis.     

Microglia Induce Neurotoxic IL-17+ γδ T Cells Dependent on TLR2, TLR4, and TLR9 Activation

Background

Interleukin-17 (IL-17) acts as a key regulator in central nervous system (CNS) inflammation. γδ T cells are an important innate source of IL-17. Both IL-17+ γδ T cells and microglia, the major resident immune cells of the brain, are involved in various CNS disorders such as multiple sclerosis and stroke. Also, activation of Toll-like receptor (TLR) signaling pathways contributes to CNS damage. However, the mechanisms underlying the regulation and interaction of these cellular and molecular components remain unclear.

Objective

In this study, we investigated the crosstalk between γδ T cells and microglia activated by TLRs in the context of neuronal damage. To this end, co-cultures of IL-17+ γδ T cells, neurons, and microglia were analyzed by immunocytochemistry, flow cytometry, ELISA and multiplex immunoassays.

Results

We report here that IL-17+ γδ T cells but not naïve γδ T cells induce a dose- and time-dependent decrease of neuronal viability in vitro. While direct stimulation of γδ T cells with various TLR ligands did not result in up-regulation of CD69, CD25, or in IL-17 secretion, supernatants of microglia stimulated by ligands specific for TLR2, TLR4, TLR7, or TLR9 induced activation of γδ T cells through IL-1β and IL-23, as indicated by up-regulation of CD69 and CD25 and by secretion of vast amounts of IL-17. This effect was dependent on the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) expressed by both γδ T cells and microglia, but did not require the expression of TLRs by γδ T cells. Similarly to cytokine-primed IL-17+ γδ T cells, IL-17+ γδ T cells induced by supernatants derived from TLR-activated microglia also caused neurotoxicity in vitro. While these neurotoxic effects required stimulation of TLR2, TLR4, or TLR9 in microglia, neuronal injury mediated by bone marrow-derived macrophages did not require TLR signaling. Neurotoxicity mediated by IL-17+ γδ T cells required a direct cell-cell contact between T cells and neurons.

Conclusion

Taken together, these results point to a crucial role for microglia activated through TLRs in polarization of γδ T cells towards neurotoxic IL-17+ γδ T cells.     

Keratan sulfate expression in microglia is diminished in the spinal cord in experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is an animal model of Guillain–Barré syndrome, an inflammatory demyelination disease of the peripheral nervous system. Although this disease has been extensively studied on peripheral nerves, the pathology of the central nervous system has not been fully understood. Previous studies demonstrate that expression of keratan sulfate (KS), the sugar chain of proteoglycan, is associated with activated microglia/macrophages accumulated after neuronal injuries. Unexpectedly, we found here that KS is rather diminished in rat EAN. KS was restrictively expressed in microglia in the spinal cord of normal rats. KS was positive in 50% microglia in the ventral horn and 20% in the dorsal horn. In EAN, microglia increased in number and expressed the activation marker CD68, but KS expression was abolished. Concomitantly, pro-inflammatory cytokines, i.e., interferon (IFN)-γ, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α, were increased in the spinal cord of EAN rats, whereas anti-inflammatory cytokines, such as IL-4 and IL-10, were decreased. In addition, silencing of KSGal6ST attenuated KS expression on the primary cultured microglia and upregulated expression of some activation markers (TNF-α, IL-1β, and iNOS) under the stimulation with lipopolysaccharide and IFN-γ. This study demonstrates for the first time a close association of EAN and disappearance of KS on microglia. KS expression could be a useful marker to evaluate the status of polyneuropathy.     

Gamma Interferon (IFN-γ) Receptor Restricts Systemic Dengue Virus Replication and Prevents Paralysis in IFN-α/β Receptor-Deficient Mice

We previously reported that mice lacking alpha/beta and gamma interferon receptors (IFN-α/βR and -γR) uniformly exhibit paralysis following infection with the dengue virus (DENV) clinical isolate PL046, while only a subset of mice lacking the IFN-γR alone and virtually no mice lacking the IFN-α/βR alone develop paralysis. Here, using a mouse-passaged variant of PL046, strain S221, we show that in the absence of the IFN-α/βR, signaling through the IFN-γR confers approximately 140-fold greater resistance against systemic vascular leakage-associated dengue disease and virtually complete protection from dengue-induced paralysis. Viral replication in the spleen was assessed by immunohistochemistry and flow cytometry, which revealed a reduction in the number of infected cells due to IFN-γR signaling by 2 days after infection, coincident with elevated levels of IFN-γ in the spleen and serum. By 4 days after infection, IFN-γR signaling was found to restrict DENV replication systemically. Clearance of DENV, on the other hand, occurred in the absence of IFN-γR, except in the central nervous system (CNS) (brain and spinal cord), where clearance relied on IFN-γ from CD8⁺ T cells. These results demonstrate the roles of IFN-γR signaling in protection from initial systemic and subsequent CNS disease following DENV infection and demonstrate the importance of CD8⁺ T cells in preventing DENV-induced CNS disease.     

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.     

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.     

Interleukin-1β and Interleukin-1 Receptor Antagonist Appear in Grey Matter Additionally to White Matter Lesions during Experimental Multiple Sclerosis

Background

Multiple sclerosis (MS) has been mainly attributed to white matter (WM) pathology. However, recent evidence indicated the presence of grey matter (GM) lesions. One of the principal mediators of inflammatory processes is interleukin-1β (IL-1β), which is known to play a role in MS pathogenesis. It is unknown whether IL-1β is solely present in WM or also in GM lesions. Using an experimental MS model, we questioned whether IL-1β and the IL-1 receptor antagonist (IL-1ra) are present in GM in addition to affected WM regions.

Methods

The expression of IL-1β and IL-1ra in chronic-relapsing EAE (cr-EAE) rats was examined using in situ hybridization, immunohistochemistry and real-time PCR. Rats were sacrificed at the peak of the first disease phase, the trough of the remission phase, and at the peak of the relapse. Histopathological characteristics of CNS lesions were studied using immunohistochemistry for PLP, CD68 and CD3 and Oil-Red O histochemistry.

Results

IL-1β and IL-ra expression appears to a similar extent in affected GM and WM regions in the brain and spinal cord of cr-EAE rats, particularly in perivascular and periventricular locations. IL-1β and IL-1ra expression was dedicated to macrophages and/or activated microglial cells, at sites of starting demyelination. The time-dependent expression of IL-1β and IL-1ra revealed that within the spinal cord IL-1β and IL-1ra mRNA remained present throughout the disease, whereas in the brain their expression disappeared during the relapse.

Conclusions

The appearance of IL-1β expressing cells in GM within the CNS during cr-EAE may explain the occurrence of several clinical deficits present in EAE and MS which cannot be attributed solely to the presence of IL-1β in WM. Endogenously produced IL-1ra seems not capable to counteract IL-1β-induced effects. We put forward that IL-1β may behold promise as a target to address GM, in addition to WM, related pathology in MS.     

IFN-β Inhibits the Increased Expression of IL-9 during Experimental Autoimmune Uveoretinitis

Purpose

It has been shown that IL-9 plays a proinflammatory role in the pathogenesis of certain autoimmune diseases. This study was designed to investigate the possible role of IL-9 in the development of experimental autoimmune uveoretinitis (EAU) and the effect of IFN-β on its expression.

Methods

EAU was induced in B10RIII mice by immunization with interphotoreceptor retinoid-binding protein peptide 161–180 (IRBP_161–180). IFN-β was administered subcutaneously to IRBP_161–180 immunized mice every other day from day one before immunization to the end of the study. Splenocytes and draining lymph node (DLN) cells from EAU mice or control mice or EAU mice treated with IFN-β or PBS were stimulated with anti-CD3/CD28 or IRBP_161–180 for 3 days. Naïve T cells cultured under Th1 or Th17 polarizing conditions were incubated in the presence or absence of IFN-β for 4 days. Effector/memory T cells were activated by anti-CD3/CD28 in the presence or absence of IFN-β for 3 days. IFN-β-treated monocytes were cocultured with naïve T cells or effector/memory T cells for 3 days. Culture supernatants were collected and IL-9 was detected by ELISA.

Results

IL-9 expression in splenocytes and DLN cells was increased in EAU mice during the inflammatory phase and returned back to lower levels during the recovery phase. IFN-β in vivo treatment significantly inhibited EAU activity in association with a down-regulated expression of IL-9. In vitro polarized Th1 and Th17 cells both secreted IL-9 and the addition of IFN-β suppressed production of IL-9 by both Th subsets. Beside its effect on polarized Th cells, IFN-β also suppressed the secretion of IL-9 by effector/memory T cells. However, IFN-β-treated monocytes had no effect on the production of IL-9 when cocultured with naïve or effector/memory T cells.

Conclusion

IL-9 expression is increased during EAU which could be suppressed by IFN-β.     

The Aldosterone-Mineralocorticoid Receptor Pathway Exerts Anti-Inflammatory Effects in Endotoxin-Induced Uveitis

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.