Eugene D. Ponomarev

Modulation of the cannabinoid CB2 receptor in microglial cells in response to inflammatory stimuli

The cannabinoid system is known to be important in neuronal regulation, but is also capable of modulating immune function. Although the CNS resident microglial cells have been shown to express the CB2 subtype of cannabinoid receptor during non‐immune‐mediated pathological conditions, little is known about the expression of the cannabinoid system during immune‐mediated CNS pathology. To examine this question, we measured CB2 receptor mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) and, by real‐time PCR, found a 100‐fold increase in CB2 receptor mRNA expression during EAE onset. We next determined whether microglial cells specifically express the CB2 receptor during EAE, and found that activated microglial cells expressed 10‐fold more CB2 receptor than microglia in the resting state. To determine the signals required for the up‐regulation of the CB2 receptor, we cultured microglial cells with combinations of γ‐interferon (IFN‐γ) and granulocyte) macrophage‐colony stimulating factor (GM‐CSF), which both promote microglial cell activation and are expressed in the CNS during EAE, and found that they synergized, resulting in an eight to 10‐fold increase in the CB2 receptor. We found no difference in the amount of the CB2 receptor ligand, 2‐arachidonylglycerol (2‐AG), in the spinal cord during EAE. These data demonstrate that microglial cell activation is accompanied by CB2 receptor up‐regulation, suggesting that this receptor plays an important role in microglial cell function in the CNS during autoimmune‐induced inflammation.

CNS-Derived Interleukin-4 Is Essential for the Regulation of Autoimmune Inflammation and Induces a State of Alternative Activation in Microglial Cells

Regulation of inflammation in the CNS is essential to prevent irreversible cellular damage that can occur in neurodegenerative diseases such as multiple sclerosis (MS). We investigated the role of interleukin-4 (IL-4) in regulating CNS inflammation using the animal model of MS, experimental autoimmune encephalomyelitis (EAE). We found that CNS-derived IL-4 was a critical regulator because mice with a deficiency in IL-4 production in the CNS, but not the periphery, had exacerbated EAE associated with a significant increase in the absolute number of infiltrating inflammatory cells. We also found that CNS-resident microglial cells in both the resting and activated state produced the protein Ym1, which is a marker of alternatively activated macrophages (aaMΦs), in an IL-4-dependent manner. This aaMΦ phenotype extended to the lack of nitric oxide (NO) production by activated microglial cells, which is a marker of classically activated macrophages. We also show that IL-4 induced the expression of Ym1 in peripheral infiltrating macrophages, which also produce NO. Thus, macrophages that migrate into the CNS exhibit a dual phenotype. These data indicate that IL-4 production in the CNS is essential for controlling autoimmune inflammation by inducing a microglial cell aaMΦ phenotype. Macrophages that have undergone alternative activation have been shown to be important in tissue repair; thus, our results suggest a new role for microglial cells in the regulation of inflammation in the CNS.

Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells.

The cannabinoid system is immunomodulatory and has been targeted as a treatment for the central nervous system (CNS) autoimmune disease multiple sclerosis. Using an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we investigated the role of the CB1 and CB2 cannabinoid receptors in regulating CNS autoimmunity. We found that CB1 receptor expression by neurons, but not T cells, was required for cannabinoid-mediated EAE suppression. In contrast, CB2 receptor expression by encephalitogenic T cells was critical for controlling inflammation associated with EAE. CB2-deficient T cells in the CNS during EAE exhibited reduced levels of apoptosis, a higher rate of proliferation and increased production of inflammatory cytokines, resulting in severe clinical disease. Together, our results demonstrate that the cannabinoid system within the CNS plays a critical role in regulating autoimmune inflammation, with the CNS directly suppressing T-cell effector function via the CB2 receptor.

GM-CSF production by autoreactive T cells is required for the activation of microglial cells and the onset of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a CNS autoimmune disease believed to be triggered by T cells secreting Th1-specific proinflammatory cytokines, such as GM-CSF. In the animal model of MS, experimental autoimmune encephalomyelitis (EAE), Th1 but not Th2 cells have been shown to induce disease; however, to date, no single encephalitogenic T cell-derived cytokine has been shown to be required for EAE onset. Because GM-CSF-deficient mice have been shown to be resistant to EAE following immunization with myelin self-Ag, we investigated the cellular source of the required GM-CSF and found that GM-CSF production by encephalitogenic T cells, but not CNS resident or other peripheral cells, was required for EAE induction. Furthermore, we showed that microglial cell activation, but not peripheral macrophage activation, was a GM-CSF-dependent process. Activation of microglial cells by the injection of LPS abrogated the GM-CSF requirement for EAE induction, suggesting that microglial cell activation is required for EAE onset. These data also demonstrate that GM-CSF is a critical Th1 cell-derived cytokine required for the initiation of CNS inflammation associated with EAE, and likely MS.

MicroRNAs are Universal Regulators of Differentiation, Activation, and Polarization of Microglia and Macrophages in Normal and Diseased CNS

MicroRNAs (miRNAs) are a class of small (∼22 nucleotides) noncoding RNAs involved in the regulation of gene expression at the post‐translational level. It is estimated that 30–90% of human genes are regulated by miRNAs, which makes these molecules of great importance for cell growth, activation, and differentiation. Microglia is CNS‐resident cells of a myeloid lineage that play an important role in immune surveillance and are actively involved in many neurologic pathologies. Although the exact origin of microglia remains enigmatic, it is established that primitive macrophages from a yolk sac populate the brain and spinal cord in normal conditions throughout development. During various pathological events such as neuroinflammation, bone marrow derived myeloid cells also migrate into the CNS. Within the CNS, both primitive macrophages from the yolk sac and bone marrow derived myeloid cells acquire a specific phenotype upon interaction with other cell types within the CNS microenvironment. The factors that drive differentiation of progenitors into microglia and control the state of activation of microglia and bone marrow‐derived myeloid cells within the CNS are not well understood. In this review we will summarize the role of miRNAs during activation and differentiation of myeloid cells. The role of miR‐124 in the adaptation of microglia and macrophages to the CNS microenvironment will be further discussed. We will also summarize the role of miRNAs as modulators of activation of microglia and microphages. Finally, we will describe the role of miR‐155 and miR‐124 in the polarization of macrophages towards classically and alternatively activated phenotypes.

CD40 Expression by Microglial Cells Is Required for Their Completion of a Two-Step Activation Process during Central Nervous System Autoimmune Inflammation

Microglial cells are monocytic lineage cells that reside in the CNS and have the capacity to become activated during various pathological conditions. Although it was demonstrated that activation of microglial cells could be achieved in vitro by the engagement of CD40-CD40L interactions in combination with proinflammatory cytokines, the exact factors that mediate activation of microglial cells in vivo during CNS autoimmunity are ill-defined. To investigate the role of CD40 in microglial cell activation during experimental autoimmune encephalomyelitis (EAE), we used bone marrow chimera mice that allowed us to distinguish microglial cells from peripheral macrophages and render microglial cells deficient in CD40. We found that the first step of microglial cell activation was CD40-independent and occurred during EAE onset. The first step of activation consisted of microglial cell proliferation and up-regulation of the activation markers MHC class II, CD40, and CD86. At the peak of disease, microglial cells underwent a second step of activation, which was characterized by a further enhancement in activation marker expression along with a reduction in proliferation. The second step of microglial cell activation was CD40-dependent and the failure of CD40-deficient microglial cells to achieve a full level of activation during EAE was correlated with reduced expansion of encephalitogenic T cells and leukocyte infiltration in the CNS, and amelioration of clinical symptoms. Thus, our findings demonstrate that CD40 expression on microglial cells is necessary to complete their activation process during EAE, which is important for disease progression.

γδ T Cells Regulate the Extent and Duration of Inflammation in the Central Nervous System by a Fas Ligand-Dependent Mechanism

γδ T cells have been shown to regulate immune responses associated with inflammation, but the mechanism of this regulation is largely unknown. Using the experimental autoimmune encephalomyelitis (EAE) model of the human CNS autoimmune disease multiple sclerosis, we demonstrate that γδ T cells are important regulators of CNS inflammation. This was shown using γδ T cell-deficient mice that were unable to recover from EAE. The chronic disease was accompanied by a prolonged presence of both macrophages and lymphocytes in the CNS. This extended inflammatory response was due to alterations in both cell proliferation and death. In mice lacking γδ T cells, proliferation of encephalitogenic T cells was 3-fold higher, and caspase activity, indicating apoptosis, was 2-fold lower compared with those in control mice recovering from EAE. γδ T cell-deficient mice reconstituted with wild-type γδ T cells recovered from EAE and resolved inflammation in the CNS, whereas mice reconstituted with Fas ligand-dysfunctional γδ T cells did not. Thus, γδ T cells regulate both inflammation in the CNS and disease recovery via Fas/Fas ligand-induced apoptosis of encephalitogenic T cells, and a quick resolution of inflammation in the CNS is essential to prevent permanent damage to the CNS resulting in chronic disease.

IL-4/IL-13-Dependent and Independent Expression of miR-124 and Its Contribution to M2 Phenotype of Monocytic Cells in Normal Conditions and during Allergic Inflammation

Monocytic cells exhibit a high level of heterogeneity and have two distinct modes of their activation: 1) classical M1 path associated with inflammation and tissue damage, and 2) alternative M2 path. Although it has been demonstrated that M2 macrophages play an important role in the regulation of the allergic immune responses, tissue maintenance and repair, little is known about the mechanisms that determine the M2 phenotype. We have previously shown that miR-124 is expressed in microglia that exhibit the M2 phenotype and overexpression of miR-124 in macrophages resulted in downregulation of a number of M1 markers (MHC class II, CD86) and up-regulation of several M2 markers (Fizz1, Arg1). We further investigated whether the polarization of macrophages towards the M2 phenotype induced miR-124 expression. We found that exposure of cells to IL-4 and IL-13 resulted in the upregulation of miR-124 in macrophages. We also demonstrated that IL-4 induced expression of three miR-124 precursor transcripts with predominant expression of pri-miR-124.3, suggesting regulation of miR-124 expression by IL-4 on a transcriptional level. Expression of miR-124 in microglia did not depend on IL-4 and/or IL-13, whereas expression of miR-124 in lung resident macrophages was IL-4 and IL-13-dependent and was upregulated by systemic administration of IL-4 or during allergic inflammation. Upregulation of several M2 markers (CD206, Ym1) and downregulation of the M1 markers (CD86, iNOS, TNF) in M2-polarized macrophages was abrogated by a miR-124 inhibitor, suggesting that this microRNA contributed to the M2 phenotype development and maintenance. Finally we showed that human CD14+CD16+ intermediate monocytes, which are found in increased numbers in patients with allergies and bronchial asthma, expressed high levels of miR-124 and exhibited other properties of M2-like cells. Thus, our study suggests that miR-124 serves as a regulator of the M2 polarization in various subsets of monocytic cells both in vitro and in vivo.

γδ T Cell Regulation of IFN-γ Production by Central Nervous System-Infiltrating Encephalitogenic T Cells: Correlation with Recovery from Experimental Autoimmune Encephalomyelitis

Interferon-γ has been shown to be important for the resolution of inflammation associated with CNS autoimmunity. Because one of the roles of γδ T cells is the regulation of inflammation, we asked whether γδ T cells were able to regulate CNS inflammation using the autoimmune disease mouse model experimental autoimmune encephalomyelitis (EAE). We show that the presence of γδ T cells was needed to promote the production of IFN-γ by both CD4 and CD8 T cells in the CNS before the onset of EAE. This regulation was shown to be independent of the ability of γδ T cells to produce IFN-γ, and was specific to T cells in the CNS, as no alterations in IFN-γ production were detectable in γδ T cell-deficient mice in the spleen and lymph nodes of mice with EAE or following immunization. Analysis of TCRγδ gene usage in the CNS showed that the only TCRδ V gene families present in the CNS before EAE onset are from the DV7s6 and DV105s1 gene families. We also show that the primary IFN-γ-producing cells in the CNS are the encephalitogenic T cells, and that γδ T cell-deficient mice are unable to resolve EAE disease symptoms like control mice, thus exhibiting a long-term chronic disease course similar to that observed in IFN-γ-deficient mice. These data suggest that CNS resident γδ T cells promote the production of IFN-γ by encephalitogenic T cells in the CNS, which is ultimately required for the recovery from EAE.

Spontaneous apoptosis and expression of cell surface heat-shock proteins in cultured EL-4 lymphoma cells

Abstract. The expression of heat‐shock proteins (HSPs) is enhanced in stressed cells and can protect cells from stress‐induced injury. However, existing data about the relationship between apoptosis and HSP expression is contradictory. In this paper, a mouse lymphoma cell death model system is used to detect simultaneously both the process of apoptosis and the level of HSP expression. The model was established after discovering that spontaneous apoptosis and spontaneous cell surface HSP expression occurs in EL‐4 mouse lymphoma cells during normal optimal culture conditions. The data show that apoptotic EL‐4 cells had higher levels of hsp25, hsp60, hsp70 and hsp90 exposed on the plasma membrane surface than viable cells. The level of surface HSPs was found to increase through several stages of early and late apoptotic death as measured by flow cytometry, with the highest levels observed during the loss of cell membrane phospholipid asymmetry. Heat shock and actinomycin D significantly increased the proportion of apoptotic cells in culture. However, hyperthermia only stimulated a weak and temporary increase in surface HSP expression, whereas actinomycin D strongly elevated the level of surface and intracellular HSPs, particularly in live cells. These results show an associative relationship between apoptosis and HSP expression. The relationship between the progression of cell death and HSP expression suggests a role for membrane HSP expression in programmed cell death.