The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF.

Interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) require exposure to IL-23 to become encephalitogenic, but the mechanism by which IL-23 promotes their pathogenicity is not known. Here we found that IL-23 induced production of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) in T(H)17 cells and that GM-CSF had an essential role in their encephalitogenicity. Our findings identify a chief mechanism that underlies the important role of IL-23 in autoimmune diseases. IL-23 induced a positive feedback loop whereby GM-CSF secreted by T(H)17 cells stimulated the production of IL-23 by antigen-presenting cells. Such cross-regulation of IL-23 and GM-CSF explains the similar pattern of resistance to autoimmunity when either of the two cytokines is absent and identifies T(H)17 cells as a crucial source of GM-CSF in autoimmune inflammation.

Myeloperoxidase as a Marker to Differentiate Mouse Monocyte/Macrophage Subsets.

Macrophages are present in every tissue in the body and play essential roles in homeostasis and host defense against microorganisms. Some tissue macrophages derive from the yolk sac/fetal liver that populate tissues for life. Other tissue macrophages derive from monocytes that differentiate in the bone marrow and circulate through tissues via the blood and lymphatics. Circulating monocytes are very plastic and differentiate into macrophages with specialized functions upon entering tissues. Specialized monocyte/macrophage subsets have been difficult to differentiate based on cell surface markers. Here, using a combination of "pan" monocyte/macrophage markers and flow cytometry, we asked whether myeloperoxidase (MPO) could be used as a marker of pro-inflammatory monocyte/macrophage subsets. MPO is of interest because of its potent microbicidal activity. In wild-type SPF housed mice, we found that MPO+ monocytes/macrophages were present in peripheral blood, spleen, small and large intestines, and mesenteric lymph nodes, but not the central nervous system. Only monocytes/macrophages that expressed cell surface F4/80 and/or Ly6C co-expressed MPO with the highest expression in F4/80HiLy6CHi subsets regardless of tissue. These cumulative data indicate that MPO expression can be used as an additional marker to differentiate between monocyte/macrophage subsets with pro-inflammatory and microbicidal activity in a variety of tissues.

Cannabinoid CB2 receptors in the mouse brain: relevance for Alzheimer's disease.

BACKGROUND: Because of their low levels of expression and the inadequacy of current research tools, CB2 cannabinoid receptors (CB2R) have been difficult to study, particularly in the brain. This receptor is especially relevant in the context of neuroinflammation, so novel tools are needed to unveil its pathophysiological role(s). METHODS: We have generated a transgenic mouse model in which the expression of enhanced green fluorescent protein (EGFP) is under the control of the cnr2 gene promoter through the insertion of an Internal Ribosomal Entry Site followed by the EGFP coding region immediately 3' of the cnr2 gene and crossed these mice with mice expressing five familial Alzheimer's disease (AD) mutations (5xFAD). RESULTS: Expression of EGFP in control mice was below the level of detection in all regions of the central nervous system (CNS) that we examined. CB2R-dependent-EGFP expression was detected in the CNS of 3-month-old AD mice in areas of intense inflammation and amyloid deposition; expression was coincident with the appearance of plaques in the cortex, hippocampus, brain stem, and thalamus. The expression of EGFP increased as a function of plaque formation and subsequent microgliosis and was restricted to microglial cells located in close proximity to neuritic plaques. AD mice with CB2R deletion exhibited decreased neuritic plaques with no changes in IL1ß expression. CONCLUSIONS: Using a novel reporter mouse line, we found no evidence for CB2R expression in the healthy CNS but clear up-regulation in the context of amyloid-triggered neuroinflammation. Data from CB2R null mice indicate that they play a complex role in the response to plaque formation.

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.

Myeloperoxidase: A new player in autoimmunity.

Myeloperoxidase (MPO) is the most toxic enzyme found in the azurophilic granules of neutrophils. MPO utilizes H2O2 to generate hypochlorous acid (HClO) and other reactive moieties, which kill pathogens during infections. In contrast, in the setting of sterile inflammation, MPO and MPO-derived oxidants are thought to be pathogenic, promoting inflammation and causing tissue damage. In contrast, evidence also exists that MPO can limit the extent of immune responses. Elevated MPO levels and activity are observed in a number of autoimmune diseases including in the central nervous system (CNS) of multiple sclerosis (MS) and the joints of rheumatoid arthritis (RA) patients. A pathogenic role for MPO in driving autoimmune inflammation was demonstrated using mouse models. Mechanisms whereby MPO is thought to contribute to disease pathogenesis include tuning of adaptive immune responses and/or the induction of vascular permeability.

Gut Microbial Dysbiosis Due to Helicobacter Drives an Increase in Marginal Zone B Cells in the Absence of IL-10 Signaling in Macrophages.

It is clear that IL-10 plays an essential role in maintaining homeostasis in the gut in response to the microbiome. However, it is unknown whether IL-10 also facilitates immune homeostasis at distal sites. To address this question, we asked whether splenic immune populations were altered in IL-10-deficient (Il10(-/-)) mice in which differences in animal husbandry history were associated with susceptibility to spontaneous enterocolitis that is microbiome dependent. The susceptible mice exhibited a significant increase in splenic macrophages, neutrophils, and marginal zone (MZ) B cells that was inhibited by IL-10 signaling in myeloid, but not B cells. The increase in macrophages was due to increased proliferation that correlated with a subsequent enhancement in MZ B cell differentiation. Cohousing and antibiotic treatment studies suggested that the alteration in immune homeostasis in the spleen was microbiome dependent. The 16S rRNA sequencing revealed that susceptible mice harbored a different microbiome with a significant increase in the abundance of the bacterial genus Helicobacter. The introduction of Helicobacter hepaticus to the gut of nonsusceptible mice was sufficient to drive macrophage expansion and MZ B cell development. Given that myeloid cells and MZ B cells are part of the first line of defense against blood-borne pathogens, their increase following a breach in the gut epithelial barrier would be protective. Thus, IL-10 is an essential gatekeeper that maintains immune homeostasis at distal sites that can become functionally imbalanced upon the introduction of specific pathogenic bacteria to the intestinal track.

Inhibition of myeloperoxidase at the peak of experimental autoimmune encephalomyelitis restores blood-brain barrier integrity and ameliorates disease severity.

Oxidative stress is thought to contribute to disease pathogenesis in the central nervous system (CNS) disease multiple sclerosis (MS). Myeloperoxidase (MPO), a potent peroxidase that generates toxic radicals and oxidants, is increased in the CNS during MS. However, the exact mechanism whereby MPO drives MS pathology is not known. We addressed this question by inhibiting MPO in mice with experimental autoimmune encephalomyelitis (EAE) using our non-toxic MPO inhibitor N-acetyl lysyltyrosylcysteine amide (KYC). We found that therapeutic administration of KYC for 5 days starting at the peak of disease significantly attenuated EAE disease severity, reduced myeloid cell numbers and permeability of the blood-brain barrier. These data indicate that inhibition of MPO by KYC restores blood-brain barrier integrity thereby limiting migration of myeloid cells into the CNS that drive EAE pathogenesis. In addition, these observations indicate that KYC may be an effective therapeutic agent for the treatment of MS. We propose that during experimental autoimmune encephalomyelitis (EAE) onset macrophages and neutrophils migrate into the CNS and upon activation release myeloperoxidase (MPO) that promotes disruption of the blood-brain barrier (BBB) and disease progression. KYC restores BBB function by inhibiting MPO activity and in so doing ameliorates disease progression.

Tumor Suppressor Interferon-Regulatory Factor 1 Counteracts the Germinal Center Reaction Driven by a Cancer-Associated Gammaherpesvirus.

UNLABELLED: Gammaherpesviruses are ubiquitous pathogens that are associated with the development of B cell lymphomas. Gammaherpesviruses employ multiple mechanisms to transiently stimulate a broad, polyclonal germinal center reaction, an inherently mutagenic stage of B cell differentiation that is thought to be the primary target of malignant transformation in virus-driven lymphomagenesis. We found that this gammaherpesvirus-driven germinal center expansion was exaggerated and lost its transient nature in the absence of interferon-regulatory factor 1 (IRF-1), a transcription factor with antiviral and tumor suppressor functions. Uncontrolled and persistent expansion of germinal center B cells led to pathological changes in the spleens of chronically infected IRF-1-deficient animals. Additionally, we found decreased IRF-1 expression in cases of human posttransplant lymphoproliferative disorder, a malignant condition associated with gammaherpesvirus infection. The results of our study define an unappreciated role for IRF-1 in B cell biology and provide insight into the potential mechanism of gammaherpesvirus-driven lymphomagenesis. IMPORTANCE: Gammaherpesviruses establish lifelong infection in most adults and are associated with B cell lymphomas. While the infection is asymptomatic in many hosts, it is critical to identify individuals who may be at an increased risk of virus-induced cancer. Such identification is currently impossible, as the host risk factors that predispose individuals toward viral lymphomagenesis are poorly understood. The current study identifies interferon-regulatory factor 1 (IRF-1) to be one of such candidate host factors. Specifically, we found that IRF-1 enforces long-term suppression of an inherently mutagenic stage of B cell differentiation that gammaherpesviruses are thought to target for transformation. Correspondingly, in the absence of IRF-1, chronic gammaherpesvirus infection induced pathological changes in the spleens of infected animals. Further, we found decreased IRF-1 expression in human gammaherpesvirus-induced B cell malignancies.

IL-10-independent regulatory B-cell subsets and mechanisms of action.

Although classically B cells are known to play important roles in immune protection via humoral immunity, recently their regulatory mechanisms have been best appreciated in the context of autoimmunity. Several studies have identified different subsets of regulatory B cells that vary not only in their phenotype but also in their mechanism of action. Although the best-studied mechanism of B-cell immune regulation is IL-10 production, other IL-10-independent mechanisms have been proposed. These include maintenance of CD4(+)Foxp3(+) regulatory T cells; production of transforming growth factor-ß, IL-35, IgM or adenosine or expression of PD-L1 (programmed death 1 ligand 1) or FasL (Fas ligand). Given that B-cell-targeted therapy is being increasingly used in the clinic, a complete understanding of the mechanisms whereby B cells regulate inflammation associated with specific diseases is required for designing safe and effective immunotherapies targeting B cells.

An increase in tolerogenic dendritic cell and natural regulatory T cell numbers during experimental autoimmune encephalomyelitis in Rras-/- mice results in attenuated disease.

R-Ras is a member of the Ras superfamily of small GTPases, which are regulators of various cellular processes, including adhesion, survival, proliferation, trafficking, and cytokine production. R-Ras is expressed by immune cells and has been shown to modulate dendritic cell (DC) function in vitro and has been associated with liver autoimmunity. We used Rras-deficient mice to study the mechanism whereby R-Ras contributes to autoimmunity using experimental autoimmune encephalomyelitis (EAE), a mouse model of the CNS autoimmune disease multiple sclerosis. We found that a lack of R-Ras in peripheral immune cells resulted in attenuated EAE disease. Further investigation revealed that, during EAE, absence of R-Ras promoted the formation of MHC II(low) DC concomitant with a significant increase in proliferation of natural regulatory T cells, resulting in an increase in their cell numbers in the periphery. Our study suggests a novel role for R-Ras in promoting autoimmunity through negative regulation of natural regulatory T cell numbers by inhibiting the development of MHCII(low) DC with tolerogenic potential.

Interrelatedness between dysbiosis in the gut microbiota due to immunodeficiency and disease penetrance of colitis.

The composition of the microbiome in health and disease has only recently become a major research focus. Although it is clear that an imbalance or dysbiosis in the microbiota is associated with disease, its interrelatedness to disease penetrance is largely unknown. Inflammatory bowel disease (IBD) is an excellent disease in which to explore these questions because of the extensive genetic studies identifying disease susceptibility loci and the ability to easily sample the intestinal microbiota in IBD patients due to the accessibility of stool samples. In addition, mouse models of IBD have contributed to our understanding of the interrelatedness of the gut microbiota and genes associated with IBD. The power of the mouse studies is that multiple colitis models exist that can be used in combination with genetically modified mice that harbour deficiencies in IBD susceptibility genes. Collectively, these studies revealed that bacterial dysbiosis does occur in human IBD and in mouse colitis models. In addition, with an emphasis on immune genes, the mouse studies provided evidence that specific immune regulatory proteins associated with IBD influence the gut microbiota in a manner consistent with disease penetrance. In this review, we will discuss studies in both humans and mice that demonstrate the impact of immunodeficiences in interleukin-10, interleukin-17, nucleotide-binding oligomerization domain (NOD) 2, NOD-like receptor proteins 3 and 6, Toll-like receptor or IgA have on the interrelatedness between the composition of the gut microbiota and disease penetrance of IBD and its mouse models.

A novel IL-10-independent regulatory role for B cells in suppressing autoimmunity by maintenance of regulatory T cells via GITR ligand.

B cells are important for the regulation of autoimmune responses. In experimental autoimmune encephalomyelitis (EAE), B cells are required for spontaneous recovery in acute models. Production of IL-10 by regulatory B cells has been shown to modulate the severity EAE and other autoimmune diseases. Previously, we suggested that B cells regulated the number of CD4(+)Foxp3(+) T regulatory cells (Treg) in the CNS during EAE. Because Treg suppress autoimmune responses, we asked whether B cells control autoimmunity by maintenance of Treg numbers. B cell deficiency achieved either genetically (µMT) or by depletion with anti-CD20 resulted in a significant reduction in the number of peripheral but not thymic Treg. Adoptive transfer of WT B cells into µMT mice restored both Treg numbers and recovery from EAE. When we investigated the mechanism whereby B cells induce the proliferation of Treg and EAE recovery, we found that glucocorticoid-induced TNF ligand, but not IL-10, expression by B cells was required. Of clinical significance is the finding that anti-CD20 depletion of B cells accelerated spontaneous EAE and colitis. Our results demonstrate that B cells play a major role in immune tolerance required for the prevention of autoimmunity by maintenance of Treg via their expression of glucocorticoid-induced TNFR ligand.

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 CB(1) and CB(2) cannabinoid receptors in regulating CNS autoimmunity. We found that CB(1) receptor expression by neurons, but not T cells, was required for cannabinoid-mediated EAE suppression. In contrast, CB(2) receptor expression by encephalitogenic T cells was critical for controlling inflammation associated with EAE. CB(2)-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 CB(2) receptor.

Ingested (oral) adrenocorticotropic hormone (ACTH) inhibits interleukin-17 in the central nervous system after adoptive transfer of T helper (Th)1/Th17 T cells in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system (CNS) that resembles multiple sclerosis (MS) and provides a useful animal model for the evaluation of mechanisms of action for potential immunomodulatory therapies. We have previously shown that oral adrenocorticotropic hormone (ACTH) decreased either interleukin (IL)-17 and/or interferon (IFN)γ in the CNS during EAE. OBJECTIVE: We wanted to examine whether oral ACTH showed a preferential effect on Th17 as opposed to Th1 phenotypes. DESIGN/METHODS: We therefore examined whether oral ACTH could inhibit EAE in the C57BL/6 (B6) mouse strain after adoptive transfer of equal quantities of Th17 (CD4+IL-17+) and Th1 (CD4+IFN-γ+) T cells generated after in vitro skewing. B6 mice were injected with a 1:1 ratio of Th1:Th17 T cells and were gavaged daily with control scrambled peptide (s-MSH) or 10 µg ACTH. RESULTS: Ingested (oral) ACTH attenuated ongoing clinical EAE disease and decreased the frequencies of Th17 cells in the spleen and in the CNS, but not Th1. CONCLUSIONS: These findings suggest that there was preferential regulation of Th17 cells by oral ACTH compared to Th1 T cells in the CNS.

Cannabinoid receptor 2 is critical for the homing and retention of marginal zone B lineage cells and for efficient T-independent immune responses.

The endocannabinoid system has emerged as an important regulator of immune responses, with the cannabinoid receptor 2 (CB2) and its principle ligand 2-archidonoylglycerol playing a major role. How CB2 regulates B cell functions is not clear, even though they express the highest levels of CB2 among immune cell subsets. In this study, we show that CB2-deficient mice have a significant reduction in the absolute number of marginal zone (MZ) B cells and their immediate precursor, transitional-2 MZ precursor. The loss of MZ lineage cells in CB2(-/-) mice was shown to be B cell intrinsic using bone marrow chimeras and was not due to a developmental or functional defect as determined by B cell phenotype, proliferation, and Ig production. Furthermore, CB2(-/-) B cells were similar to wild type in their apoptosis, cell turnover, and BCR and Notch-2 signaling. We then demonstrated that CB2(-/-) MZ lineage B cells were less efficient at homing to the MZ and that their subsequent retention was also regulated by CB2. CB2(-/-) mice immunized with T-independent Ags produced significantly less Ag-specific IgM. This study demonstrates that CB2 positively regulates T-independent immune responses by controlling the localization and positioning of MZ lineage cells to the MZ.

Ingested (Oral) Adrenocorticotropic Hormone Inhibits IL-17 in the Central Nervous System in the Mouse Model of Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the CNS that resembles multiple sclerosis and provides a useful animal model for the evaluation of mechanisms of action for potential immunomodulatory therapies. We have previously shown that oral adrenocorticotropic hormone (ACTH) decreased IL-17 in the gut lamina propria and the spleen and increased CD4+ Foxp3+ T regulatory cells and IL-10 in the spleen during EAE in the C57BL/6 mouse. However, we did not investigate the specific cellular alterations of proinflammatory and anti-inflammatory factors in the CNS. The aim was to determine if oral ACTH would have a similar clinical effect on inflammatory cytokines in the gut and define specific cellular effects in the CNS in an alternative strain of mice. SJL/J mice were immunized with proteolipid protein peptide 138-151 and gavaged with scrambled ACTH (scrambled α-melanocyte-stimulating hormone) or ACTH 1-39 during ongoing disease. Ingested (oral) ACTH attenuated ongoing clinical EAE disease, decreased IL-6 production, and increased T regulatory cells in the lamina propria and decreased CD4+ and γδ IL-17 production in the CNS. Ingested ACTH attenuated EAE clinical disease by decreasing IL-6 in the gut-associated lymphoid tissue and decreasing IL-17 in the CNS.

Targeting transmembrane-domain-less MOG expression to platelets prevents disease development in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system with no cure yet. Here, we report genetic engineering of hematopoietic stem cells (HSCs) to express myelin oligodendrocyte glycoprotein (MOG), specifically in platelets, as a means of intervention to induce immune tolerance in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. The platelet-specific αIIb promoter was used to drive either a full-length or truncated MOG expression cassette. Platelet-MOG expression was introduced by lentivirus transduction of HSCs followed by transplantation. MOG protein was detected on the cell surface of platelets only in full-length MOG-transduced recipients, but MOG was detected in transmembrane-domain-less MOG1-157-transduced platelets intracellularly. We found that targeting MOG expression to platelets could prevent EAE development and attenuate disease severity, including the loss of bladder control in transduced recipients. Elimination of the transmembrane domains of MOG significantly enhanced the clinical efficacy in preventing the onset and development of the disease and induced CD4+Foxp3+ Treg cells in the EAE model. Together, our data demonstrated that targeting transmembrane domain-deleted MOG expression to platelets is an effective strategy to induce immune tolerance in EAE, which could be a promising approach for the treatment of patients with MS autoimmune disease.

Characterization of the Cell Surface Phenotype and Regulatory Activity of B-Cell IgD Low (BDL) Regulatory B Cells.

B-cell IgD Low (BDL) B cells have been shown to promote immunological tolerance by inducing proliferation of CD4+Foxp3+ T-regulatory cells (Treg) in a glucocorticoid-induced tumor necrosis factor receptor-related protein ligand (GITRL, Tnfsf18)-dependent manner. BDL cells constitute a small subset of splenic B lymphocytes that, in mice, are characterized by the B220+IgMintCD21intCD23+CD93-IgDlow/- cell surface expression profile. In this chapter, we show the flow cytometry gating strategy developed to identify and purify BDL. In addition, we describe an in vitro assay and two in vivo assays to assess BDL regulatory activity by quantitating Treg expansion/proliferation and indicate how they can be used in mouse models of disease. Collectively, these methods are useful to track and quantitate BDL and Treg numbers and assess their regulatory activity in inflammatory disease models.