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.

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.

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.

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.

Myeloperoxidase Inhibition Ameliorates Plaque Psoriasis in Mice.

Plaque psoriasis is a common inflammatory condition of the skin characterized by red, flaking lesions. Current therapies for plaque psoriasis target many facets of the autoimmune response, but there is an incomplete understanding of how oxidative damage produced by enzymes such as myeloperoxidase contributes to skin pathology. In this study, we used the Aldara (Imiquimod) cream model of plaque psoriasis in mice to assess myeloperoxidase inhibition for treating psoriatic skin lesions. To assess skin inflammation severity, an innovative mouse psoriasis scoring system was developed. We found that myeloperoxidase inhibition ameliorated psoriasis severity when administered either systemically or topically. The findings of this study support the role of oxidative damage in plaque psoriasis pathology and present potential new therapeutic avenues for further exploration.

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.

Comparison of the Efficacy and Safety of Anti-CD20 B Cells Depleting Drugs in Multiple Sclerosis.

Rituximab, ocrelizumab, ofatumumab and ublituximab are disease modifying therapies (DMT) currently used in the treatment of multiple sclerosis (MS) or are in advanced stages of clinical trials. These monoclonal antibodies deplete B cells by targeting the cell surface protein CD20. This review highlights the similarities and major differences between the four agents. We summarize data from various clinical trials of each of these therapeutics and discuss their efficacy and safety. Additional considerations regarding the route of administration and cost are presented. Among the four therapeutics, only ocrelizumab is approved for primary progressive (PP) MS. Infusion/injection related reactions (IRRs) are the most common adverse events associated with all four therapeutics. In phase III trials of ocrelizumab and ofatumumab, the incidence of IRRs was lower with ofatumumab. Ofatumumab is unique among the four therapeutics due to its availability as a subcutaneous injection (SQ). Although SQ administration may be appealing for some patients it may raise concerns regarding medication compliance among physicians. Phase II trials studying ublituximab for the treatment of RMS yielded promising results. Phase III trials are currently comparing the efficacy of ublituximab to teriflunomide.

Discovery and Function of B-Cell IgD Low (BDL) B Cells in Immune Tolerance.

It is now appreciated that in addition to their role in humoral immunity, B cells also exert regulatory mechanisms that lead to attenuation of inflammatory responses. The concept of B-cell regulation became well recognized when mice deficient in B cells due to genetic disruption were shown to be refractory to recovery from the signs of experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. This seminal study spurred the search for B-cell regulatory phenotypes and mechanisms of action. Our approach was to utilize differential B-cell depletion with anti-CD20 to retain B cells whose presence were required to achieve EAE recovery. Utilizing flow cytometry, adoptive cell therapy and genetic approaches, we discovered a new B-cell subset that, upon adoptive transfer into B cell-deficient mice, was sufficient to promote EAE recovery. This B-cell subset is IgM+, but due to low/negative IgD cell surface expression, it was named B-cell IgD low (BDL). Mechanistically, we found that in the absence of BDL, the absolute cell number of CD4+Foxp3+ T regulatory cells (Treg), essential for immune tolerance, was significantly reduced. Furthermore, we found that BDL expression of glucocorticoid-induced tumor necrosis factor ligand (GITRL) was essential for induction of Treg proliferation and maintenance of their homeostasis. Thus, we have identified a new B-cell subset that is critical for immunological tolerance through interactions with Treg.

Characterization of Definitive Regulatory B Cell Subsets by Cell Surface Phenotype, Function and Context.

Regulatory B cell or "Breg" is a broad term that represents the anti-inflammatory activity of B cells, but does not describe their individual phenotypes, specific mechanisms of regulation or relevant disease contexts. Thus, given the variety of B cell regulatory mechanisms reported in human disease and their animal models, a more thorough and comprehensive identification strategy is needed for tracking and comparing B cell subsets between research groups and in clinical settings. This review summarizes the discovery process and mechanism of action for well-defined regulatory B cell subsets with an emphasis on the mouse model of multiple sclerosis experimental autoimmune encephalomyelitis. We discuss the importance of conducting thorough B cell phenotyping along with mechanistic studies prior to defining a particular subset of B cells as Breg. Since virtually all B cell subsets can exert regulatory activity, it is timely for their definitive identification across studies.

Neutrophil-Derived Myeloperoxidase Facilitates Both the Induction and Elicitation Phases of Contact Hypersensitivity.

Background: Allergic contact dermatitis (ACD) is a common skin disorder affecting an estimated 15-20% of the general population. The mouse model of ACD is contact hypersensitivity (CHS), which consists of two phases: induction and elicitation. Although neutrophils are required for both CHS disease phases their mechanisms of action are poorly understood. Neutrophils release myeloperoxidase (MPO) that through oxidation of biomolecules leads to cellular damage. Objectives: This study investigated mechanisms whereby MPO contributes to CHS pathogenesis. Methods: CHS was induced in mice using oxazolone (OX) as the initiating hapten applied to the skin. After 7 days, CHS was elicited by application of OX to the ear and disease severity was measured by ear thickness and vascular permeability in the ear. The role of MPO in the two phases of CHS was determined utilizing MPO-deficient mice and a specific MPO inhibitor. Results: During the CHS induction phase MPO-deficiency lead to a reduction in IL-1ß production in the skin and a subsequent reduction in migratory dendritic cells (DC) and effector T cells in the draining lymph node. During the elicitation phase, inhibition of MPO significantly reduced both ear swelling and vascular permeability. Conclusion: MPO plays dual roles in CHS pathogenesis. In the initiation phase MPO promotes IL-1ß production in the skin and activation of migratory DC that promote effector T cell priming. In the elicitation phase MPO drives vascular permeability contributing to inflammation. These results indicate that MPO it could be a potential therapeutic target for the treatment of ACD in humans.

Ingested ACTH blocks Th17 production by inhibiting GALT IL-6.

BACKGROUND: EAE is an inflammatory autoimmune process of the CNS that resembles multiple sclerosis (MS) and provides a useful animal model for the evaluation of mechanisms of action for potential immunomodulatory therapies. Oral ACTH (adrenocorticotropic hormone) can decrease clinical disease, IL-17 and Th1-like encephalitogenic IFN-γ secretion and increase Treg frequency. The mechanism by which oral ACTH decreases inflammatory proteins and increases Treg cell frequencies is unknown. OBJECTIVE: IL-6 is a pivotal cytokine in the gut that determines the relative frequencies of Th17 vs Treg cells. We examined whether oral ACTH inhibited IL-6 in the gut associated lymphoid tissue (GALT) in EAE. DESIGN/METHODS: B6 mice were immunized with MOG peptide 35-55 and gavaged with scrambled ACTH (scrambled melanocyte stimulating hormone [scrambled α-MSH]) or ACTH 1-39 during ongoing disease. RESULTS: Ingested (oral) ACTH inhibited ongoing clinical disease. In the lamina propria (LP) immune compartment, there were significantly less CD11b + IL-6 and IL-17 producing lymphocytes from ACTH fed mice compared to s-MSH fed mice. There was also a decrease in the frequency of IL-17 and IFN-γ producing spleen cells and an increase in CD4 + FoxP3+ Treg cell frequency in ACTH fed mice compared to s-MSH fed control spleens. There were less IFN-γ producing CNS lymphocytes in ACTH fed mice compared to s-MSH fed control CNS. CONCLUSIONS: Ingested ACTH inhibits EAE clinical disease by inhibiting IL-6 in the GALT.

A Novel Sex-Dependent Target for the Treatment of Postoperative Pain: The NLRP3 Inflammasome.

In recent years the innate immune system has been shown to be crucial for the pathogenesis of postoperative pain. The mediators released by innate immune cells drive the sensitization of sensory neurons following injury by directly acting on peripheral nerve terminals at the injury site. The predominate sensitization signaling pathway involves the proinflammatory cytokine interleukin-1ß (IL-1ß). IL-1ß is known to cause pain by directly acting on sensory neurons. Evidence demonstrates that blockade of IL-1ß signaling decreases postoperative pain, however complete blockade of IL-1ß signaling increases the risk of infection and decreases effective wound healing. IL-1ß requires activation by an inflammasome; inflammasomes are cytosolic receptors of the innate immune system. NOD-like receptor protein 3 (NLRP3) is the predominant inflammasome activated by endogenous molecules that are released by tissue injury such as that which occurs during neuropathic and inflammatory pain disorders. Given that selective inhibition of NLRP3 alleviates postoperative mechanical pain, its selective targeting may be a novel and effective strategy for the treatment of pain that would avoid complications of global IL-1ß inhibition. Moreover, NLRP3 is activated in pain in a sex-dependent and cell type-dependent manner. Sex differences in the innate immune system have been shown to drive pain and sensitization through different mechanisms in inflammatory and neuropathic pain disorders, indicating that it is imperative that both sexes are studied when researchers investigate and identify new targets for pain therapeutics. This review will highlight the roles of the innate immune response, the NLRP3 inflammasome, and sex differences in neuropathic and inflammatory pain.

Mature IgDlow/- B cells maintain tolerance by promoting regulatory T cell homeostasis.

A number of different B cell subsets have been shown to exhibit regulatory activity using a variety of mechanisms to attenuate inflammatory diseases. Here we show, using anti-CD20-mediated partial B cell depletion in mice, that a population of mature B cells distinguishable by IgDlow/- expression maintains tolerance by, at least in part, promoting CD4+Foxp3+ regulatory T cell homeostatic expansion via glucocorticoid-induced tumor necrosis factor receptor ligand, or GITRL. Cell surface phenotyping, transcriptome analysis and developmental study data show that B cells expressing IgD at a low level (BDL) are a novel population of mature B cells that emerge in the spleen from the transitional-2 stage paralleling the differentiation of follicular B cells. The cell surface phenotype and regulatory function of BDL are highly suggestive that they are a new B cell subset. Human splenic and peripheral blood IgDlow/- B cells also exhibit BDL regulatory activity, rendering them of therapeutic interest.

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.

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.

Mechanisms of Regulatory B cell Function in Autoimmune and Inflammatory Diseases beyond IL-10.

In the past two decades it has become clear that in addition to antigen presentation and antibody production B cells play prominent roles in immune regulation. While B cell-derived IL-10 has garnered much attention, B cells also effectively regulate inflammation by a variety of IL-10-independent mechanisms. B cell regulation has been studied in both autoimmune and inflammatory diseases. While collectively called regulatory B cells (Breg), no definitive phenotype has emerged for B cells with regulatory potential. This has made their study challenging and thus unique B cell regulatory mechanisms have emerged in a disease-dependent manner. Thus to harness the therapeutic potential of Breg, further studies are needed to understand how they emerge and are induced to evoke their regulatory activities.

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.