Katarzyna Maresz

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.

B cell regulation of CD4+CD25+ T regulatory cells and IL-10 via B7 is essential for recovery from experimental autoimmune encephalomyelitis.

CD4+CD25+ T regulatory (Treg) cells expressing the Foxp3 transcription factor have been shown to be present in the CNS during the autoimmune disease experimental autoimmune encephalomyelitis (EAE) and can inhibit EAE clinical disease by an IL-10-dependent mechanism. In addition, IL-10 expression in the CNS late in the EAE disease course has been attributed to recovery. However, it is not known how Treg cells and IL-10 expressions are regulated during EAE. We have previously shown a requirement for B cells in recovery from EAE and here investigated whether this was due to a deficiency in Treg cells and IL-10 in the CNS. We found that B cell deficiency resulted in a delay in the emergence of Foxp3-expressing Treg cells and IL-10 in the CNS during EAE, but not in the periphery. Reconstitution with wild-type B cells resulted in disease recovery and normalized IL-10 and Foxp3 expression. However, reconstitution with B7-deficient B cells did not. Furthermore, we show that IL-10 and Foxp3 expression is enhanced in CNS nonencephalitogenic T cells. These data suggest a novel mechanism whereby B cells regulate CD4+CD25+ Treg cells via B7 and subsequently enter the CNS and suppress autoimmune inflammation, mediating recovery.

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.

What we know and do not know about the cannabinoid receptor 2 (CB2)

It has been well appreciated that the endocannabinoid system can regulate immune responses via the cannabinoid receptor 2 (CB2), which is primarily expressed by cells of the hematopoietic system. The endocannabinoid system is composed of receptors, ligands and enzymes controlling the synthesis and degradation of endocannabinoids. Along with endocannabinoids, both plant-derived and synthetic cannabinoids have been shown to bind to and signal through CB2 via G proteins leading to both inhibitory and stimulatory signals depending on the biological process. Because no cannabinoid ligand has been identified that only binds to CB2, the generation of mice deficient in CB2 has greatly expanded our knowledge of how CB2 contributes to immune cell development and function in health and disease. In regards to humans, genetic studies have associated CB2 with a variety of human diseases. Here, we review the endocannabinoid system with an emphasis on CB2 and its role in the immune system.

IL-13 induces the expression of the alternative activation marker Ym1 in a subset of testicular macrophages.

Macrophages are thought to play an important role in the maintenance of immune privilege in the testis, which functions to prevent immune responses to developing sperm. Two populations of macrophages are known to exist in the testis, one of which exhibits immunosuppressive activity. Macrophages that are alternatively activated with either IL-4 or IL-13 have been shown to be anti-inflammatory and promote wound healing. Expression of the Ym1 protein is an established marker of alternatively activated macrophages. Testicular macrophages were examined for expression of Ym1 protein, and it was found to be highly expressed in a subpopulation of CD11b(+) cells. Furthermore, we have shown that Ym1 protein expression in the testis is dependent upon IL-13R signaling, and that IL-13 is produced in the testis. These data suggest that IL-13 plays a role in testicular immune privilege by the maintenance of an alternatively activated macrophage population.