Effect of the innate immune response on development of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus (TMEV) infection of susceptible mice leads to the development of demyelinating disease in the central nervous system (CNS) associated with an inflammatory immune response. The demyelinating disease in mice has similarities to multiple sclerosis in humans and is used as an experimental model for the human disease. The innate immune response initiates the immune response to TMEV through innate immune receptors on cells that recognize components of the virus and activate intracellular signaling that leads to the expression of innate immune cytokines, chemokines, and effector molecules. The innate immune response directly affects the development of the adaptive immune response, especially the T cell response, which mediates viral clearance. However, infection of Swiss Jim Laboratory (SJL) mice with TMEV leads to a persistent virus infection of the microglia/macrophage in the CNS which contributes to the development of demyelinating disease. Susceptibility to demyelinating disease has been linked to the T cell response against the virus. However, the current studies will examine the role of the innate immune response to TMEV and the affect it has on the adaptive immune response and development of demyelinating disease following TMEV infection. The innate immune cytokines, chemokines, and effector molecules as well as the innate immune cells, both CNS resident and infiltrating peripheral cells, all contribute to the innate immune response following TMEV and may affect susceptibility to demyelinating disease.

The Ifng gene is essential for Vdr gene expression and vitamin D3-mediated reduction of the pathogenic T cell burden in the central nervous system in experimental autoimmune encephalomyelitis, a multiple sclerosis model.

Compelling evidence suggests that vitamin D3 insufficiency may contribute causally to multiple sclerosis (MS) risk. Experimental autoimmune encephalomyelitis (EAE) research firmly supports this hypothesis. Vitamin D3 supports 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3) synthesis in the CNS, initiating biological processes that reduce pathogenic CD4+ T cell longevity. MS is prevalent in Sardinia despite high ambient UV irradiation, challenging the vitamin D-MS hypothesis. Sardinian MS patients frequently carry a low Ifng expresser allele, suggesting that inadequate IFN-γ may undermine vitamin D3-mediated inhibition of demyelinating disease. Testing this hypothesis, we found vitamin D3 failed to inhibit EAE in female Ifng knockout (GKO) mice, unlike wild-type mice. The two strains did not differ in Cyp27b1 and Cyp24a1 gene expression, implying equivalent vitamin D3 metabolism in the CNS. The 1,25-(OH)2D3 inhibited EAE in both strains, but 2-fold more 1,25-(OH)2D3 was needed in GKO mice, causing hypercalcemic toxicity. Unexpectedly, GKO mice had very low Vdr gene expression in the CNS. Injecting IFN-γ intracranially into adult mice did not increase Vdr gene expression. Correlating with low Vdr expression, GKO mice had more numerous pathogenic Th1 and Th17 cells in the CNS, and 1,25-(OH)2D3 reduced these cells in GKO and wild-type mice without altering Foxp3+ regulatory T cells. Thus, the Ifng gene was needed for CNS Vdr gene expression and vitamin D3-dependent mechanisms that inhibit EAE. Individuals with inadequate Ifng expression may have increased MS risk despite high ambient UV irradiation because of low Vdr gene expression and a high encephalitogenic T cell burden in the CNS.

Endoplasmic reticulum stress regulates the innate immunity critical transcription factor IRF3.

IFN regulatory factor 3 (IRF3) regulates early type I IFNs and other genes involved in innate immunity. We have previously shown that cells undergoing an endoplasmic reticulum (ER) stress response called the unfolded protein response produce synergistically augmented IFN-ß when stimulated with pattern recognition receptor agonists such as LPS. Concomitant ER stress and LPS stimulation resulted in greater recruitment of the IRF3 transcription factor to ifnb1 gene regulatory elements. In this study, we used murine cells to demonstrate that both oxygen-glucose deprivation and pharmacologic unfolded protein response inducers trigger phosphorylation and nuclear translocation of IRF3, even in the absence of exogenous LPS. Different ER stressors used distinct mechanisms to activate IRF3: IRF3 phosphorylation due to calcium-mobilizing ER stress (thapsigargin treatment, oxygen-glucose deprivation) critically depended upon stimulator of IFN gene, an ER-resident nucleic acid-responsive molecule. However, calcium mobilization alone by ionomycin was insufficient for IRF3 phosphorylation. In contrast, other forms of ER stress (e.g., tunicamycin treatment) promote IRF3 phosphorylation independently of stimulator of IFN gene and TANK-binding kinase 1. Rather, IRF3 activation by tunicamycin and 2-deoxyglucose was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine protease inhibitor that blocks activating transcription factor 6 processing. Interfering with ER stress-induced IRF3 activation abrogated IFN-ß synergy. Together, these data suggest ER stress primes cells to respond to innate immune stimuli by activating the IRF3 transcription factor. Our results also suggest certain types of ER stress accomplish IRF3 phosphorylation by co-opting existing innate immune pathogen response pathways. These data have implications for diseases involving ER stress and type I IFN.

Title: P2x7 Receptor Activation and Estrogen Status Drive Neuroinflammatory Mechanisms in a Rat Model for Dry Eye.

Dry eye disease (DED) is recognized as a chronic inflammatory condition with an increase in tear osmolarity and loss of tear film integrity. DED is often accompanied by adverse ocular symptoms which are more prevalent in females than males. The basis for ocular hyperalgesia in DED remains uncertain; however, both peripheral and central neural mechanisms are implicated. A model for aqueous deficient DED, exorbital gland excision, was used to determine if activation of the purinergic receptor subtype 7, P2X7R, expressed by non-neural cells in peripheral and central trigeminal nerve pathways, contributed to persistent ocular hyperalgesia. Densitometry of trigeminal brainstem sections revealed increases in P2X7R, the myeloid cell marker Iba1, and the inflammasome, NLRP3, of estradiol-treated DED females compared to estradiol-treated sham females, while expression in DED males and DED females not given estradiol displayed minor changes. No evidence of immune cell infiltration into the trigeminal brainstem was seen in DED rats; however, markers for microglia activation (Iba1) were increased in all groups. Isolated microglia expressed increased levels of P2X7R and P2X4R, IL-1ß (Ιnterleukin-1ß), NLRP3, and iNOS (nitric oxide synthase). Further, estradiol-treated DED females displayed greater increases in P2X7R, IL-1ß and NLRP3 expression compared to untreated DED females. Orbicularis oculi muscle activity (OOemg) evoked by ocular instillation of hypertonic saline (HS) was recorded as a surrogate measure of ocular hyperalgesia and was markedly enhanced in all DED groups compared to sham rats. Systemic minocycline reduced HS-evoked OOemg in all DED groups compared to sham rats. Local microinjection in the caudal trigeminal brainstem of an antagonist for P2X7R (A804598) greatly reduced HS-evoked OOemg activity in all DE groups, while responses in sham groups were not affected. Intra-trigeminal ganglion injection of siRNA for P2X7R significantly reduced HS-evoked OOemg activity in all DED groups, while evoked responses in sham animals were not affected. These results indicated that activation of P2X7R at central and peripheral sites in trigeminal pain pathways contributed to an increase in ocular hyperalgesia and microglia activation in DED males and females. Estrogen treatment in females further amplified ocular hyperalgesia and neuroimmune responses in this model for aqueous deficient DED.

Activation of microglia depends on Na+/H+ exchange-mediated H+ homeostasis.

H(+) extrusion is important for sustained NADPH oxidase activation after "respiratory" burst in macrophage/microglia activation. In this study, we investigated the role of Na(+)/H(+) exchanger isoform 1 (NHE-1) in activation of microglia after lipopolysaccharide (LPS) or oxygen and glucose deprivation and reoxygenation (OGD/REOX) exposure. NHE-1 functioned in maintaining basal pH(i) of immortalized M4T.4 microglia or mouse primary microglia. Pharmacological inhibition of NHE-1 activity with the potent inhibitor cariporide [HOE 642 (4-isopropyl-3-methylsulfonyl-benzoyl-guanidine-methanesulfonate)] abolished pH(i) regulation in microglia under basal conditions. Activation of microglia either by LPS, phorbol myristate acetate, or OGD/REOX accelerated pH(i) regulation and caused pH(i) elevation, which was accompanied with an increase in [Na(+)](i) and [Ca(2+)](i) as well as production of superoxide anion and cytokines. Interestingly, inhibition of NHE-1 not only abolished pH(i) regulation but also reduced production of superoxide anion as well as expression of cytokines and inducible nitric oxide synthase. Together, these results reveal that there was a concurrent activation of NHE-1 in microglia in response to proinflammatory stimuli. The study suggests that NHE-1 functions to maintain microglial pH(i) homeostasis allowing for sustained NADPH oxidase function and "respiratory" burst.

The innate immune response affects the development of the autoimmune response in Theiler's virus-induced demyelinating disease.

Multiple sclerosis (MS) is a human CNS autoimmune demyelinating disease. Epidemiological evidence has suggested a role for virus infection in the initiation and/or exacerbation of MS. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease serves as a relevant mouse model for MS. TMEV-infected mice develop a demyelinating disease with clinical symptoms beginning around 35 days after infection, which is associated with development of myelin-specific, PLP(139-151), CD4(+) T cell responses. Viruses have been suggested to initiate autoimmune disease through bystander activation of immune cells or through bystander damage to tissue during infection. We examined the effect of the innate immune response on development of autoimmune demyelinating disease by altering the innate immune response through administration of innate immune cytokines, IFN-alpha or IFN-beta, or antiserum against the type I IFNs during the innate immune response to TMEV. Administration of IFN-beta, but not IFN-alpha, to TMEV- infected mice led to reduced myelin-specific CD4(+) T cell responses and reduced demyelinating disease, which was associated with decreased immune cell infiltration into the CNS and increased expression of IL-10 in the CNS. Conversely, administration of antiserum to IFN-beta led to a more severe demyelinating disease. In addition, administration of poly(I:C), which is an innate immune agonist, to TMEV-infected mice during the innate immune response resulted in decreased myelin-specific CD4(+) T cell responses and reduced demyelinating disease. These results demonstrate that activating or enhancing the innate immune response can reduce the subsequent initiation and progression of the autoimmune response and demyelinating disease.

Innate immune CD11b+Gr-1+ cells, suppressor cells, affect the immune response during Theiler's virus-induced demyelinating disease.

Multiple sclerosis is a demyelinating disease associated with an inflammatory immune response in the CNS. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a relevant mouse model for the study of multiple sclerosis. TMEV infection of susceptible mice leads to a persistent virus infection of the CNS which contributes to development of demyelinating disease. We have previously shown that the innate immune response can affect the development and progression of demyelinating disease. In the current studies, we determined that the predominant infiltrating cells during the innate immune response are CD11b(+)Ly6C(+) cells. CD11b(+)Ly6C(+) cells are immature myeloid cells that have exited the bone marrow without maturing and have been shown to suppress CD4(+) and CD8(+) T cell responses. Therefore, we wanted to determine what role these cells play in development and progression of demyelinating disease. TMEV-infected mice depleted of CD11b(+)Ly6C(+) cells during the innate immune response developed a reduced demyelinating disease which was associated with a decreased myelin-specific CD4(+) T cell response and a decreased inflammatory immune response in the CNS. TMEV-infected mice depleted of CD11b(+)Ly6C(+) cells had increased virus-specific CD4(+) and CD8(+) T cell responses during early virus infection associated with increased expression of IFN-gamma and IL-17 and decreased expression of IL-10 in the CNS. These results suggest that CD11b(+)Ly6C(+) cells which infiltrate into the CNS during the innate immune response are myeloid-derived suppressor cells that suppress virus-specific T cell responses and contribute to the development of demyelinating disease.

Exosomes Secreted by Microglia During Virus Infection in the Central Nervous System Activate an Inflammatory Response in Bystander Cells.

Microglia become persistently infected during Theiler's murine encephalomyelitis virus (TMEV) infection in the central nervous system (CNS) of susceptible mice. We have previously shown that microglia infected with TMEV become activated through the innate immune receptors to express type I interferons, cytokines, and chemokines. Persistent TMEV infection in the CNS promotes chronic neuroinflammation and development of demyelinating disease similar to multiple sclerosis. In the current studies, we wanted to determine whether TMEV-infected microglia secrete exosomes which contribute to neuroinflammation in the CNS thus promoting the development of demyelinating disease. Exosomes are vesicles containing RNA, DNA, and proteins that are released from one cell and taken up by another cell to facilitate communication between cells. These studies isolated exosomes secreted by microglia during TMEV infection in vitro as well as exosomes secreted by microglia during early TMEV infection in mice. These studies show that microglia secrete exosomes during TMEV infection which contain the viral RNA coding region. The exosomes secreted by microglia during TMEV infection can be taken up by uninfected bystander cells, including CNS resident microglia, astrocytes, and neurons. The viral RNA in the exosomes can be transferred to the bystander cells. In addition, the bystander cells that took up these exosomes were activated through the innate immune response to express type I interferons, IFNα and IFNß, pro-inflammatory cytokines, IL-6, IL-12, and TNFα, and chemokines, CCL2. Most interestingly, exosomes secreted by microglia during early TMEV infection in mice activated an inflammatory response when transferred to the brains of naïve mice. These results show that exosomes secreted by microglia during early TMEV infection contain viral RNA and can activate uninfected bystander CNS cells to promote an inflammatory immune response. Thus, exosomes secreted by microglia during virus infection may promote viral persistence and neuroinflammation which contributes to the development of demyelinating disease.

Extracellular Vesicles Secreted by Tumor Cells Promote the Generation of Suppressive Monocytes.

Monocytes are among the first cells to infiltrate the tumor microenvironment. The conversion of monocytes to suppressor cells in the tumor microenvironment is crucial in evasion of the immune response and tumor maintenance. Tumor cells may secrete products that promote the conversion of monocytes to suppressor cells. Cells secrete extracellular vesicles (EVs) containing cargos of genetic materials and proteins as a way to communicate with neighboring cells. During pathologic conditions like cancers, tumor cells increase their EVs production containing microRNA, RNA, and proteins that may affect the immune cell response, contributing to the immunosuppressive microenvironment. Our studies show that EVs secreted by a wide range of murine tumor cells, including osteosarcoma, glioma, colon carcinoma, sarcoma, and melanoma, can be taken up by bone marrow-derived monocytes. The monocytes that took up the EVs secreted by tumor cells matured toward an immune-suppressive phenotype by upregulating the expression of suppressive cytokines and effector molecules. The monocytes also downregulated MHC class II and costimulatory molecules while increasing the expression of PD-L1 on their surface after taking up EVs from tumor cells. Most importantly, monocytes exposed to EVs secreted by tumor cells suppressed activated Ag-specific CD4+ T cells. These results show that tumor cells from several different tumor types secrete EVs which promote the conversion of monocytes into suppressor cells, thus promoting immune evasion. These studies suggest that EVs secreted by tumors are potentially a new target for future cancer therapy.

hsa-MiR-19a-3p and hsa-MiR-19b-3p Are Associated with Spinal Cord Injury-Induced Neuropathic Pain: Findings from a Genome-Wide MicroRNA Expression Profiling Screen.

Neuropathic pain in spinal cord injury (SCI) is associated with inflammation in both the peripheral and central nervous system (CNS), which may contribute to the initiation and maintenance of persistent pain. An understanding of factors contributing to neuroinflammation may lead to new therapeutic targets for neuropathic pain. Moreover, novel circulating biomarkers of neuropathic pain may facilitate earlier and more effective treatment. MicroRNAs (miRNAs) are short, non-coding single-stranded RNA that have emerged as important biomarkers and molecular mediators in physiological and pathological conditions. Using a genome-wide miRNA screening approach, we studied differential miRNA expression in plasma from 68 healthy, community-dwelling adults with and without SCI enrolled in ongoing clinical studies. We detected 2367 distinct miRNAs. Of these, 383 miRNAs were differentially expressed in acute SCI or chronic SCI versus no SCI and 71 were differentially expressed in chronic neuropathic pain versus no neuropathic pain. We selected homo sapiens (hsa)-miR-19a-3p and hsa-miR-19b-3p for additional analysis based on p-value, fold change, and their known role as regulators of neuropathic pain and neuroinflammation. Both hsa-miR-19a-3p and hsa-miR-19b-3p levels were significantly higher in those with chronic SCI and severe neuropathic pain versus those with chronic SCI and no neuropathic pain. In confirmatory studies, both hsa-miR-19a-3p and hsa-miR-19b-3p have moderate to strong discriminative ability to distinguish between those with and without pain. After adjusting for opioid use, hsa-miR-19b-3p levels were positively associated with pain interference with mood. Because hsa-miR-19 levels have been shown to change in response to exercise, folic acid, and resveratrol, these studies suggest that miRNAs are potential targets of therapeutic interventions.

The bivalent ligand MCC22 potently attenuates hyperalgesia in a mouse model of cisplatin-evoked neuropathic pain without tolerance or reward.

Cisplatin and other widely employed platinum-based anticancer agents produce chemotherapy-induced peripheral neuropathy (CIPN) that often results in pain and hyperalgesia that are difficult to manage. We investigated the efficacy of a novel bivalent ligand, MCC22, for the treatment of pain arising from CIPN. MCC22 consists of mu opioid receptor (MOR) agonist and chemokine receptor 5 (CCR5) antagonist pharmacophores connected through a 22-atom spacer and was designed to target a putative MOR-CCR5 heteromer localized in pain processing areas. Mice received once daily intraperitoneal (i.p.) injections of cisplatin (1 mg/kg) for seven days and behavior testing began 7 days later. Cisplatin produced mechanical hyperalgesia that was decreased dose-dependently by MCC22 given by intrathecal (ED50 = 0.004 pmol) or i.p. (3.07 mg/kg) routes. The decrease in hyperalgesia was associated with decreased inflammatory response by microglia in the spinal cord. Unlike morphine, MCC22 given daily for nine days did not exhibit tolerance to its analgesic effect and its characteristic antihyperalgesic activity was fully retained in morphine-tolerant mice. Furthermore, MCC22 did not alter motor function and did not exhibit rewarding properties. Given the exceptional potency of MCC22 without tolerance or reward, MCC22 has the potential to vastly improve management of chronic pain due to CIPN. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'.

Pioglitazone, a PPARγ agonist, reduces cisplatin-evoked neuropathic pain by protecting against oxidative stress.

Painful peripheral neuropathy is a dose-limiting side effect of cisplatin treatment. Using a murine model of cisplatin-induced hyperalgesia, we determined whether a PPARγ synthetic agonist, pioglitazone, attenuated the development of neuropathic pain and identified underlying mechanisms. Cisplatin produced mechanical and cold hyperalgesia and decreased electrical thresholds of Aδ and C fibers, which were attenuated by coadministration of pioglitazone (10 mg/kg, intraperitoneally [i.p.]) with cisplatin. Antihyperalgesic effects of pioglitazone were blocked by the PPARγ antagonist T0070907 (10 mg/kg, i.p.). We hypothesized that the ability of pioglitazone to reduce the accumulation of reactive oxygen species (ROS) in dorsal root ganglion (DRG) neurons contributed to its antihyperalgesic activity. Effects of cisplatin and pioglitazone on somatosensory neurons were studied on dissociated mouse DRG neurons after 24 hours in vitro. Incubation of DRG neurons with cisplatin (13 µM) for 24 hours increased the occurrence of depolarization-evoked calcium transients, and these were normalized by coincubation with pioglitazone (10 µM). Oxidative stress in DRG neurons was considered a significant contributor to cisplatin-evoked hyperalgesia because a ROS scavenger attenuated hyperalgesia and normalized the evoked calcium responses when cotreated with cisplatin. Pioglitazone increased the expression and activity of ROS-reducing enzymes in DRG and normalized cisplatin-evoked changes in oxidative stress and labeling of mitochondria with the dye MitoTracker Deep Red, indicating that the antihyperalgesic effects of pioglitazone were attributed to its antioxidant properties in DRG neurons. These data demonstrate clear benefits of broadening the use of the antidiabetic drug pioglitazone, or other PPARγ agonists, to minimize the development of cisplatin-induced painful neuropathy.

Bivalent ligand MCC22 potently attenuates nociception in a murine model of sickle cell disease.

Sickle cell disease (SCD) is a chronic inflammatory disorder accompanied by chronic pain. In addition to ongoing pain and hyperalgesia, vaso-occlusive crises-induced pain can be chronic or episodic. Because analgesics typically used to treat pain are not very effective in SCD, opioids, including morphine, are a primary treatment for managing pain in SCD but are associated with many serious side effects, including constipation, tolerance, addiction, and respiratory depression. Thus, there is a need for the development of novel treatments for pain in SCD. In this study, we used the Townes transgenic mouse model of SCD to investigate the antinociceptive efficacy of the bivalent ligand, MCC22, and compared its effectiveness with morphine. MCC22 consists of a mu-opioid receptor agonist and a chemokine receptor-5 (CCR5) antagonist that are linked through a 22-atom spacer. Our results show that intraperitoneal administration of MCC22 produced exceptionally potent dose-dependent antihyperalgesia as compared to morphine, dramatically decreased evoked responses of nociceptive dorsal horn neurons, and decreased expression of proinflammatory cytokines in the spinal cord. Moreover, tolerance did not develop to its analgesic effects after repeated administration. In view of the extraordinary potency of MCC22 without tolerance, MCC22 and similar compounds may vastly improve the management of pain associated with SCD.

Vitamin D3 alters microglia immune activation by an IL-10 dependent SOCS3 mechanism.

Microglia become activated immune cells during infection or disease in the central nervous system (CNS). However, the mechanisms that downregulate activated microglia to prevent immune-mediated damage are not completely understood. Vitamin D3 has been suggested to have immunomodulatory affects, and high levels of vitamin D3 have been correlated with a decreased risk for developing some neurological diseases. Recent studies have demonstrated the synthesis of active vitamin D3, 1,25-dihydroxyvitamin D3, within the CNS, but its cellular source and neuroprotective actions remain unknown. Therefore, we wanted to determine whether microglia can respond to vitamin D3 and whether vitamin D3 alters immune activation of microglia. We have previously shown that microglia become activated by IFNγ or LPS or by infection with virus to express pro-inflammatory cytokines, chemokines, and effector molecules. In this study, activated microglia increased the expression of the vitamin D receptor and Cyp27b1, which encodes the enzyme for converting vitamin D3 into its active form, thereby enhancing their responsiveness to vitamin D3. Most importantly, the activated microglia exposed to vitamin D3 had reduced expression of pro-inflammatory cytokines, IL-6, IL-12, and TNFα, and increased expression of IL-10. The reduction in pro-inflammatory cytokines was dependent on IL-10 induction of suppressor of cytokine signaling-3 (SOCS3). Therefore, vitamin D3 increases the expression of IL-10 creating a feedback loop via SOCS3 that downregulates the pro-inflammatory immune response by activated microglia which would likewise prevent immune mediated damage in the CNS.

Innate and adaptive immune requirements for induction of autoimmune demyelinating disease by molecular mimicry.

Molecular mimicry is the main postulated mechanism by which infectious agents induce autoimmune disease. A number of animal models have been utilized to establish a link between molecular mimicry and autoimmunity. However, a model of infectious disease whereby a natural pathogen expressing a known mimic epitope can induce autoimmunity to a known self-antigen leading to clinical autoimmune disease is still lacking. We have engineered a recombinant Theiler's murine encephalomyelitis virus (TMEV) to express an encephalitogenic myelin proteolipid protein PLP139-151 epitope (PLP-TMEV) and a PLP139-151 mimic peptide naturally expressed by Haemophilus influenzae (HI-TMEV). Infection of mice with either PLP-TMEV or HI-TMEV induces early-onset disease that is associated with the activation of cross-reactive PLP139-151-specific immunopathologic CD4+ Th1 cells. Based on results from this model, we hypothesize, due to the considerable degeneracy in the T cell repertoire, that induction of full-blown autoimmune disease via molecular mimicry is a tightly regulated process requiring multiple factors related to the pathogen expressing the potential mimic epitope. In this review, we will discuss how various factors related to the infectious environment control whether or not autoimmune disease is initiated. Contributing factors include the nature of the innate immune response to the pathogen which determines the immunopathologic potential of the induced cross-reactive T cells, the capacity of the mimic epitope to be processed and presented from its natural flanking sequences in the pathogen-encoded protein, the site(s) of the primary infection in the host and the ability of the pathogen to persist, and the potential requirement for multiple infections with the same or different pathogens.

Epitope spreading and molecular mimicry as triggers of autoimmunity in the Theiler's virus-induced demyelinating disease model of multiple sclerosis.

The pathogenesis of multiple sclerosis (MS), a human demyelinating disease of the central nervous system (CNS), is currently unknown. It is widely thought that MS is an autoimmune disease which is supported by animal studies showing that myelin-specific CD4+ T cells can induce similar clinical disease in mice as observed in MS. However, the mechanism(s) of activation of these autoreactive CD4+ T cells are unknown. Although genetic susceptibility is important, other factors may be involved. Viral infections have long thought to be involved in the pathogenesis of MS although there exists little or no direct evidence implicating a role for a specific virus in MS pathogenesis. This review will discuss two models of virus-induced CNS autoimmunity, molecular mimicry and epitope spreading. These two mechanisms of activation of autoreactive T cells are presented in the context of MS.

Efficient technique for immortalization of murine microglial cells relevant for studies in murine models of multiple sclerosis.

Microglia are macrophage-like cells that populate the central nervous system (CNS) and become activated upon injury or infection. Microglia have been implicated as playing critical roles in various CNS diseases including multiple sclerosis (MS), a human autoimmune demyelinating disease, as well as in other neurodegenerative diseases. Two well-characterized models of MS, relapsing experimental autoimmune encephalomyelitis (R-EAE) and Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, are inducible in SJL mice and model the relapsing-remitting and chronic-progressive forms of MS, respectively. These models are useful for the study of the mechanisms of initiation, progression, and therapy of the disease. Currently, a major limitation to studying the functions of microglia in these murine models of MS is the restricted number of cells capable of being isolated from the CNS of neonatal mice and propagated in culture. The current studies describe the preparation of SV-40 large T antigen-immortalized mouse microglia lines, M4T.4 and M4T.6, from the SJL/J mice. The immortalization technique was very efficient requiring only 6 weeks to develop long-term, highly replicating cell lines. The resulting microglia cell lines remain quiescent, but are induced to express various immune cytokines and to function as efficient antigen presenting cells upon activation with IFN-gamma or infection with TMEV. Thus, the SV-40 large T antigen immortalized microglia lines react to innate and infectious stimuli similar to primary microglia isolated from neonatal mice, but are more easily maintained in culture. This technique should allow for the efficient cultivation of large numbers of microglial cells from a variety of disease-relevant mouse strains, including knock-out and transgenic mice.

Mouse models of multiple sclerosis: experimental autoimmune encephalomyelitis and Theiler's virus-induced demyelinating disease.

Experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalitis virus-induced demyelinating disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS). Like MS, both are characterized by mononuclear cell infiltrate into the central nervous system and demyelination. EAE is induced by either the administration of protein or peptide in adjuvant or by the adoptive transfer of encephalitogenic T-cell blasts into naïve recipients. The relative merits of each of these protocols are compared. Depending on the type of question asked, different mouse strains and peptides are used. Different disease courses are observed with different strains and different peptides in active EAE. These variations are addressed, and grading of mice in EAE is discussed. In addition to EAE induction, useful references for other disease indicators, such as delayed-type hypersensitivity, in vitro proliferation, and immunohistochemistry, are provided. TMEV-IDD is a useful model for understanding the potential viral etiology of MS. This chapter provides detailed information on the preparation of viral stocks and subsequent intracerebral infection of mice. In addition, virus plaque assay and disease assessment are discussed. Recombinant TMEV strains have been created for the study of molecular mimicry; these strains incorporate 30 various amino acid myelin epitopes within the leader region of TMEV.

IFNγ influences type I interferon response and susceptibility to Theiler's virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease in susceptible SJL mice that has similarities to multiple sclerosis in humans. TMEV infection of susceptible mice leads to a persistent virus infection of the central nervous system (CNS), which promotes the development of demyelinating disease associated with an inflammatory immune response in the CNS. TMEV infection of resistant C57BL6 mice results in viral clearance without development of demyelinating disease. Interestingly, TMEV infection of resistant mice deficient in IFNγ leads to a persistent virus infection in the CNS and development of demyelinating disease. We have previously shown that the innate immune response affects development of TMEV- induced demyelinating disease, thus we wanted to determine the role of IFNγ during the innate immune response. TMEV-infected IFNγ-deficient mice had an altered innate immune response, including reduced expression of innate immune cytokines, especially type I interferons. Administration of type I interferons, IFNα and IFNß, to TMEV-infected IFNγ-deficient mice during the innate immune response restored the expression of innate immune cytokines. Most importantly, administration of type I interferons to IFNγ-deficient mice during the innate immune response decreased the virus load in the CNS and decreased development of demyelinating disease. Microglia are the CNS resident immune cells that express innate immune receptors. In TMEV-infected IFNγ-deficient mice, microglia had reduced expression of innate immune cytokines, and administration of type I interferons to these mice restored the innate immune response by microglia. In the absence of IFNγ, microglia from TMEV-infected mice had reduced expression of some innate immune receptors and signaling molecules, especially IRF1. These results suggest that IFNγ plays an important role in the innate immune response to TMEV by enhancing the expression of innate immune cytokines, especially type I interferons, which directly affects the development of demyelinating disease.

Immune responses of microglia in the spinal cord: contribution to pain states.

The role of microglia and their contribution to the development and maintenance of pain states has emerged as an attractive field of study. Sensitization of central nociceptors and interneurons is thought to be responsible for the symptoms of chronic neuropathic pain states. Microglia interact with these neurons at the site of injury or disease as well as remotely. Microglia can be activated by phagocytosis or through the activation of a number of constitutively expressed cell surface molecules. Once activated, microglia participate in both innate and adaptive immune responses and remain active indefinitely. Activated microglia contribute to pain states through the production of pro-inflammatory cytokines, chemokines and extracellular proteases. Activated microglia also exhibit a modulated cell surface receptor and ion channel profile. The activation of several intracellular pathways in microglia has also been implicated in pain states. Attenuation of microglia activity is being presented as a viable therapeutic approach with regard to not only the reduction of pain symptoms but also in preventing the development of chronic pain states.