ATM-dependent spontaneous regression of early Eµ-myc-induced murine B-cell leukemia depends on natural killer and T cells.

Mechanisms of spontaneous tumor regression have been difficult to characterize in a systematic manner due to their rare occurrence and the lack of model systems. Here, we provide evidence that early-stage B cells in Eµ-myc mice are tumorigenic and sharply regress in the periphery between 41 and 65 days of age. Regression depended on CD4(+), CD8(+), NK1.1(+) cells and the activation of the DNA damage response, which has been shown to provide an early barrier against cancer. The DNA damage response can induce ligands that enhance immune recognition. Blockade of DNAM-1, a receptor for one such ligand, impaired tumor regression. Hence, Eµ-myc mice provide a model to study spontaneous regression and possible mechanisms of immune evasion or suppression by cancer cells.

Development of experimental autoimmune encephalomyelitis critically depends on CD137 ligand signaling.

Multiple sclerosis (MS) is a degenerative autoimmune disease of the CNS. Experimental autoimmune encephalomyelitis (EAE) is a commonly used murine model for MS. Here we report that CD137 ligand (CD137L, 4-1BB ligand, TNFS9), a member of the TNF superfamily, is critical for the development of EAE. EAE symptoms were significantly ameliorated in CD137L(-/-) mice. In the absence of CD137L, myelin oligodendrocyte glycoprotein (MOG)-specific T-cells secreted lower levels of T(h)1/T(h)17 cell-associated cytokines. MOG-specific T-cells also trafficked less efficiently to the CNS in CD137L(-/-) mice, possibly as a consequence of reduced expression of vascular cell adhesion molecule-1 (VCAM-1), which regulates leukocyte extravasation. Thus, CD137L regulates many functions of MOG-specific T-cells that contribute to EAE and may represent a novel therapeutic target for the treatment of MS.

CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species.

CD137 (4-1BB, TNFRSF9), a member of the tumor necrosis factor (TNF) receptor family, is a potent T cell co-stimulatory molecule. CD137 ligand (CD137L) is expressed by antigen presenting cells (APC) as a transmembrane protein and transmits activating signals into APC. In this study we investigated the effects of CD137L signaling in microglia, the resident APC in the central nervous system. In vitro, the murine microglia cell lines BV-2 and N9, as well as primary murine microglia responded with activation as evidenced by adherence and secretion of proinflammatory cytokines, MMP-9, and soluble intercellular adhesion molecule (ICAM). CD137L signaling is also important for microglia activation in vivo, since CD137L-deficient mice exhibited profoundly less microglia activation during experimental autoimmune encephalomyelitis (EAE) which is a well-established murine model for neuroinflammation and human multiple sclerosis (MS). Also CD137 is expressed in the CNS of mice during EAE. Activated microglia has been reported to mediate the destruction of axonal myelin sheaths and cause the death of oligodendrocytes, the main pathogenic mechanisms in EAE and MS. Corresponding to the lower microglia activation there were also fewer apoptotic oligodendrocytes in the CNS of CD137L-deficient mice. In vitro co-culture confirmed that CD137L-activated microglia induces apoptosis in oligodendrocytes, and identified reactive oxygen species as the mechanism of apoptosis induction. These data demonstrate activating effects of CD137L signaling to microglia, and show for the first time that the CD137 receptor/ligand system may be a mediator of neuroinflammatory and neurodegenerative disease, by activating microglia which in turn kill oligodendrocytes.

Generation and characterization of a novel recombinant antibody against 15-ketocholestane isolated by phage-display.

The employment of monoclonal antibodies (Mabs) to identify disease-associated biomarkers in clinical samples represents the underlying principle for many diagnostic tests. To date, these have been principally developed for protein targets with few reported applications for lipids due to their hydrophobicity and poor immunogenicity. Oxysterols represent a family of lipids implicated in diverse human diseases where Mab-based detection assays could have a profound effect on their utility as clinical biomarkers. These are usually identified in patients' samples by mass- spectrometry based approaches. Here, we describe an antibody phage-library based screening methodology for generating a recombinant monoclonal antibody (RAb) targeting the oxysterol-15-ketocholestane (15-KA), a lipid implicated in multiple sclerosis and Autoimmune Encephalomyelitis (EAE). The antibody is highly specific for 15-KA and shows little or no binding activity for other closely related oxysterols. We employ RAb2E9 to address the controversy over whether 15-KA is a true biomarker for MS/EAE and show that 15-KA is undetectable in serum taken from mice with EAE using antibody based detection methodologies; a finding confirmed by mass-spectrometry analysis. This study demonstrates the technical feasibility of using phage display to isolate highly specific antibodies against poorly immunogenic, small molecule lipids.

Damage control: how HIV survives the editor APOBEC3G.

The antiviral factor APOBEC3G upregulates the expression of ligands for the activating receptor NKG2D via DNA damage induced by the viral protein Vpr in cells infected with human immunodeficiency virus. The virus overcomes greater susceptibility to natural killer cell­mediated lysis by targeting APOBEC3G for degradation.

Suppression of experimental autoimmune encephalomyelitis by ghrelin.

Ghrelin is a recently identified gastric hormone that displays strong growth hormone-releasing activity mediated by the growth hormone secretagogue receptor. While this unique endogenous peptide participates in the regulation of energy homeostasis, increases food intake, and decreases energy expenditure, its ability to inhibit the production of proinflammatory cytokines in vitro indicates its role in the regulation of inflammatory process in vivo. Here we examine the effect of exogenous ghrelin on the development of experimental autoimmune encephalomyelitis (EAE), a representative model of multiple sclerosis. In the C57BL/6 mouse model of EAE induced by sensitization to myelin oligodendrocyte glycoprotein 35-55 peptide, we found that alternate-day s.c. injections of ghrelin (5 mug/kg/day) from day 1 to 35 significantly reduced the clinical severity of EAE. The suppression of EAE was accompanied by reduced mRNA levels of proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 in the spinal cord cellular infiltrates and microglia from ghrelin-treated mice at the peak of disease, suggesting the role of ghrelin as an antiinflammatory hormone. Consistently, ghrelin significantly suppressed the production of proinflammatory cytokines in LPS-stimulated microglia in vitro. These results shed light on the new role of ghrelin in the regulation of inflammation with possible implications for management of human diseases.

Back to the future for multiple sclerosis therapy: focus on current and emerging disease-modifying therapeutic strategies.

The last decade has seen numerous advances in the treatment of multiple sclerosis with six immunotherapeutic agents licensed for use. Although these therapeutic agents have powerful effects upon the inflammatory phase of disease, they have limitations in treating the progression of disability and in their safety profile. This review focuses on our current understanding of first- and second-line treatments for multiple sclerosis, including combination therapies, and also discusses the most promising novel therapeutic strategies on the horizon. Such agents include orally administered immunosuppressive drugs, monoclonal antibodies, antigen-specific tolerance, and neural protection and repair strategies. The challenge ahead lies in the delivery of potent drugs to inhibit inflammation and neurodegeneration while limiting side effects. Further elucidation of the pathophysiology of disease may provide new clinical targets and disease-relevant biomarkers that, in combination with proteomics, may help personalize treatment to individual patients.

NKT cell-dependent amelioration of a mouse model of multiple sclerosis by altering gut flora.

Improved hygiene has been suggested to influence certain autoimmune disorders, such as multiple sclerosis. In this study, we addressed whether altering the composition of gut flora may affect susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We administered a mixture of non-absorbing antibiotics, kanamycin, colistin, and vancomycin (KCV), orally to mice induced to develop EAE. The antibiotic treatment, beginning 1 week prior to sensitization, altered the composition of gut flora and, intriguingly, also ameliorated the development of EAE. While this result was associated with a reduced production of pro-inflammatory cytokines from the draining lymph node cells, a reduction of mesenteric Th17 cells was found to correlate with disease suppression. In addition, we found that Valpha14 invariant NKT (iNKT) cells were necessary for maintaining the mesenteric Th17 cells. The homologous effects of KCV treatment and iNKT cell depletion led us to speculate that KCV treatment may suppress EAE by altering the function of iNKT cells. Consistent with this hypothesis, KCV treatment did not suppress EAE that was induced in iNKT cell-deficient mice, although it was efficacious in mice that lacked Valpha19 mucosal-associated invariant T cells. Thus, gut flora may influence the development of EAE in a way that is dependent on iNKT cells, which has significant implications for the prevention and treatment of autoimmune diseases.

Cannabinoid-mediated neuroprotection, not immunosuppression, may be more relevant to multiple sclerosis.

Cannabinoids may exhibit symptom control in multiple sclerosis (MS). We show here that cannabinoid receptor (CBR) agonists can also be immunosuppressive and neuroprotective in models of MS. Immunosuppression was associated with reduced: myelin-specific T cell responses; central nervous system infiltration and reduced clinical disease. This was found to be largely CB(1)R-dependent and only occurred at doses that induced significant cannabimimetic effects that would not be achieved clinically. Lower, non-immunosuppressive doses of cannabinoids however, slowed the accumulation of nerve loss and disability, despite failing to inhibit relapses. This further highlights the neuroprotective potential of cannabinoids to slow the progression of MS.

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.

Cross-reactivity between peptide mimics of the immunodominant myelin proteolipid protein epitope PLP139-151: comparison of peptide priming in CFA vs. viral delivery.

Epidemiological evidence suggests that pathogens may trigger the development of autoimmune diseases such as multiple sclerosis (MS). Pathogens may trigger disease via molecular mimicry, wherein T cells generated against foreign epitopes are also cross-reactive with self-epitopes. Five pathogen-derived molecular mimics of PLP(139-151) (the immunodominant CD4(+) T cell myelin epitope in SJL mice) were previously identified. This study examines the degree of cross-reactivity between the different mimics, comparing mice primed with mimic peptide in CFA with mice infected with recombinant mimic-expressing viruses. The pattern of in vitro reactivity and ability to induce CNS disease differs between peptide priming and virus infection.

Structural requirements for initiation of cross-reactivity and CNS autoimmunity with a PLP139-151 mimic peptide derived from murine hepatitis virus.

MS is an autoimmune CNS demyelinating disease in which infection appears to be an important pathogenic factor. Molecular mimicry, the cross-activation of autoreactive T cells by mimic peptides from infectious agents, is a possible explanation for infection-induced autoimmunity. Infection of mice with a non-pathogenic strain of Theiler's murine encephalomyelitis virus (TMEV) engineered to express an epitope from Haemophilus influenzae (HI) sharing 6/13 amino acids with the dominant proteolipid protein (PLP) epitope, PLP139-151, can induce CNS autoimmune disease. Here we demonstrate that another PLP139-151 mimic sequence derived from murine hepatitis virus (MHV) which shares only 3/13 amino acids with PLP139-151 can also induce CNS autoimmune disease, but only when delivered by genetically engineered TMEV, not by immunization with the MHV peptide. Further, we demonstrate the importance of proline at the secondary MHC class II contact residue for effective cross-reactivity, as addition of this amino acid to the native MHV sequence increases its ability to cross-activate PLP139-151-specific autoreactive T cells, while substitution of proline in the HI mimic peptide has the opposite effect. This study describes a structural requirement for potential PLP139-151 mimic peptides, and provides further evidence for infection-induced molecular mimicry in the pathogenesis of autoimmune disease.

Invariant V(alpha)19i T cells regulate autoimmune inflammation.

T cells expressing an invariant V(alpha)19-J(alpha)33 T cell receptor alpha-chain (V(alpha)19i TCR) are restricted by the nonpolymorphic major histocompatibility complex class Ib molecule MR1. Whether V(alpha)19i T cells are involved in autoimmunity is not understood. Here we demonstrate that T cells expressing the V(alpha)19i TCR transgene inhibited the induction and progression of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Similarly, EAE was exacerbated in MR1-deficient mice, which lack V(alpha)19i T cells. EAE suppression was accompanied by reduced production of inflammatory mediators and increased secretion of interleukin 10. Interleukin 10 production occurred at least in part through interactions between B cells and V(alpha)19i T cells mediated by the ICOS costimulatory molecule. These results suggest an immunoregulatory function for V(alpha)19i T cells.

Effects of cannabinoid treatment on Chagas disease pathogenesis: balancing inhibition of parasite invasion and immunosuppression.

Trypanosoma cruzi invades heart cells via a calcium-dependent, G protein-mediated mechanism, leading to severe cardiac inflammation considered by some to be autoimmune in nature. Cannabinoids inhibit calcium flux and G protein signalling; as potent immunosuppressive agents, they are effective in the treatment of autoimmune disease but contraindicated for the treatment of infections. We compared the action of the synthetic cannabinoid R(+)WIN55,212 and its inactive isomer S(-)WIN55,212 on cardiac myoblast invasion: R(+)WIN55,212 inhibited invasion by over 85%. We then tested for efficacy in modulating pathogenesis in mice by assaying parasite burden in heart and blood, cellular and humoral immunity to parasite and self antigens, and mortality. R(+)WIN55,212 significantly reduced cardiac inflammation but led to considerably increased parasitaemia. Cardiac parasitosis and mortality were not significantly different in treatment and control groups. We conclude that cannabinoids can block cardiac cell puncture repair mechanisms, thereby inhibiting trypanosome invasion as predicted by the mode of drug action, but, also inhibit immune cell effector functions, offsetting the benefit of inhibition parasite cell invasion. Refined use of cannabinoids may prove therapeutic in the future, but our results raise concern about the effect of cannabis use on those chronically infected by T. cruzi and on heart cell homeostasis generally.

Cannabinoids and the immune system: potential for the treatment of inflammatory diseases?

Since the discovery of the cannabinoid receptors and their endogenous ligands, significant advances have been made in studying the physiological function of the endocannabinoid system. The presence of cannabinoid receptors on cells of the immune system and anecdotal and historical evidence suggesting that cannabis use has potent immuno-modulatory effects, has led to research directed at understanding the function and role of these receptors within the context of immunological cellular function. Studies from chronic cannabis smokers have provided much of the evidence for immunomodulatory effects of cannabis in humans, and animal and in vitro studies of immune cells such as T cells and macrophages have also provided important evidence. Cannabinoids can modulate both the function and secretion of cytokines from immune cells. Therefore, cannabinoids may be considered for treatment of inflammatory disease. This review article will highlight recent research on cannabinoids and how they interact with the immune system and also their potential use as therapeutic agents for a number of inflammatory disorders.

Initiation and exacerbation of autoimmune demyelination of the central nervous system via virus-induced molecular mimicry: implications for the pathogenesis of multiple sclerosis.

Epidemiological studies indicate that infectious agents are important in the pathogenesis of multiple sclerosis (MS). Our previous reports showed that the infection of SJL mice with a nonpathogenic variant of Theiler's murine encephalomyelitis virus (TMEV) engineered to express a naturally occurring Haemophilus influenzae-encoded molecular mimic (HI574-586) of an immunodominant self-myelin proteolipid protein epitope (PLP139-151) induced a rapid-onset demyelinating disease associated with the activation of PLP139-151-specific Th1 responses. The current results extend our previous findings in four critical respects. We show that disease initiation by the H. influenzae mimic is prevented by tolerance to the self PLP139-151 epitope, definitively proving the occurrence of infection-induced molecular mimicry. We demonstrate that the H. influenzae mimic epitope can be processed from the flanking sequences within the native mimic protein. We show that the H. influenzae mimic epitope only induces an immunopathologic self-reactive Th1 response and subsequent clinical disease in the context of the TMEV infection and not when administered in complete Freund's adjuvant, indicating that molecular mimicry-induced disease initiation requires virus-activated innate immune signals. Lastly, we show that the infection of SJL mice with TMEV expressing the H. influenzae mimic can exacerbate a previously established nonprogressive autoimmune disease of the central nervous system. Collectively, these findings illustrate the evolving mechanisms by which virus infections may contribute to both the initiation and exacerbation of autoimmune diseases, and they have important implications for MS pathogenesis.

Autoimmune tolerance eliminates relapses but fails to halt progression in a model of multiple sclerosis.

To date there has been poor translation of immunotherapies from rodent models to treatment of progressive multiple sclerosis (MS). In the robust, relapsing Biozzi ABH mouse model of MS, using a combination of a transient deletion of T cells followed by intravenous (i.v.) myelin antigen administration, established relapsing disease in EAE can be effectively silenced. However, when treatment was initiated in late stage chronic-relapsing disease, despite inhibition of further relapses, mice demonstrated evidence of disease progression shown by a deterioration in mobility and development of spasticity and indicates that targeting relapsing, immunological components of MS alone is unlikely to be sufficient to control progression in the late stages of MS.

Citrullination of central nervous system proteins during the development of experimental autoimmune encephalomyelitis.

Immunization of mammals with central nervous system (CNS)-derived proteins or peptides induces experimental autoimmune encephalomyelitis (EAE), a disease resembling the human autoimmune disease multiple sclerosis (MS). Both diseases are accompanied by destruction of a part of the of the myelin sheaths, which surround neurites in the CNS. Previous studies in MS have described alterations in the citrullination of myelin basic protein, one of the main protein constituents of the myelin sheath. Here, we show that, also during the development of EAE in mice, hypercitrullination occurs in the areas of the spinal cord that show the highest degree of inflammation and that myelin basic protein and glial fibrillary acidic protein are among the hypercitrullinated proteins. We conclude that hypercitrullination of myelin proteins in the CNS is a common phenomenon in demyelinating disease. Hypercitrullination may cause conformational changes in proteins, so the affected proteins may be involved in the pathogenesis of CNS autoimmune disease by acting as autoreactive T-cell epitopes. This is the first report in which hypercitrullination of CNS proteins in EAE is described and in which proteins other than myelin basic protein are reported to be citrullinated during autoimmune-mediated CNS inflammation.

Viral delivery of an epitope from Haemophilus influenzae induces central nervous system autoimmune disease by molecular mimicry.

Multiple sclerosis (MS) is an autoimmune CNS demyelinating disease in which infection may be an important initiating factor. Pathogen-induced cross-activation of autoimmune T cells may occur by molecular mimicry. Infection with wild-type Theiler's murine encephalomyelitis virus induces a late-onset, progressive T cell-mediated demyelinating disease, similar to MS. To determine the potential of virus-induced autoimmunity by molecular mimicry, a nonpathogenic neurotropic Theiler's murine encephalomyelitis virus variant was engineered to encode a mimic peptide from protease IV of Haemophilus influenzae (HI), sharing 6 of 13 aa with the dominant encephalitogenic proteolipid protein (PLP) epitope PLP(139-151). Infection of SJL mice with the HI mimic-expressing virus induced a rapid-onset, nonprogressive paralytic disease characterized by potent activation of self-reactive PLP(139-151)-specific CD4(+) Th1 responses. In contrast, mice immunized with the HI mimic-peptide in CFA did not develop disease, associated with the failure to induce activation of PLP(139-151)-specific CD4(+) Th1 cells. However, preinfection with the mimic-expressing virus before mimic-peptide immunization led to severe disease. Therefore, infection with a mimic-expressing virus directly initiates organ-specific T cell-mediated autoimmunity, suggesting that pathogen-delivered innate immune signals may play a crucial role in triggering differentiation of pathogenic self-reactive responses. These results have important implications for explaining the pathogenesis of MS and other autoimmune diseases.

Towards cannabis and cannabinoid treatment of multiple sclerosis.

Multiple sclerosis is a common human demyelinating disease of the central nervous system (CNS), and it is thought to involve autoimmune responses to CNS myelin antigens. Current symptomatic therapies for multiple sclerosis are in some cases ineffective and may have a high risk of serious side effects. This has led some multiple sclerosis patients to self-medicate with cannabis, which anecdotal evidence suggests may be beneficial in controlling symptoms such as spasticity, pain, tremor and bladder dysfunction. In support of these claims, results from experimental studies have suggested that cannabinoid-based treatments may be beneficial in a wide number of diseases. Furthermore, recent research in animal models of multiple sclerosis has demonstrated the efficacy of cannabinoids in controlling disease-induced symptoms such as spasticity and tremor, as well as in ameliorating the severity of clinical disease. However, these initially promising results have not yet been fully translated into the clinic. Although cannabinoid treatment of multiple sclerosis symptoms has been shown to be both well tolerated and effective in a number of subjective tests in several small-scale clinical trials, objective measures demonstrating the efficacy of cannabinoids are still lacking. Currently, a number of large-scale phase III clinical trials are under way to further elucidate the use of cannabinoids in the symptomatic treatment of multiple sclerosis. This review highlights the recent advances in our understanding of the endocannabinoid system, discusses both the experimental and clinical evidence for the use of cannabinoids to treat multiple sclerosis and explores possible future strategies of cannabinoid therapy in multiple sclerosis.