Lipoic Acid Stimulates cAMP Production in Healthy Control and Secondary Progressive MS Subjects.

Lipoic acid (LA) exhibits antioxidant and anti-inflammatory properties; supplementation reduces disease severity and T lymphocyte migration into the central nervous system in a murine model of multiple sclerosis (MS), and administration in secondary progressive MS (SPMS) subjects reduces brain atrophy compared to placebo. The mechanism of action (MOA) of LA's efficacy in suppression of MS pathology is incompletely understood. LA stimulates production of the immunomodulator cyclic AMP (cAMP) in vitro. To determine whether cAMP could be involved in the MOA of LA in vivo, we performed a clinical trial to examine whether LA stimulates cAMP production in healthy control and MS subjects, and whether there are differences in the bioavailability of LA between groups. We administered 1200 mg of oral LA to healthy control, relapsing remitting MS (RRMS) and SPMS subjects, and measured plasma LA and cAMP levels in peripheral blood mononuclear cells (PBMCs). There were no significant differences between the groups in pharmacokinetic (PK) parameters. Healthy and SPMS subjects had increased cAMP at 2 and 4 h post-LA treatment compared to baseline, while RRMS subjects showed decreases in cAMP. Additionally, plasma concentrations of prostaglandin E2 (PGE2, a known cAMP stimulator) were significantly lower in female RRMS subjects compared to female HC and SPMS subjects 4 h after LA ingestion. These data indicate that cAMP could be part of the MOA of LA in SPMS, and that there is a divergent response to LA in RRMS subjects that may have implications in the efficacy of immunomodulatory drugs. This clinical trial, "Defining the Anti-inflammatory Role of Lipoic Acid in Multiple Sclerosis," NCT00997438, is registered at https://clinicaltrials.gov/ct2/show/record/NCT00997438 .

Donor variability may mask dimethyl fumarate's effects on nuclear factor E2-related factor 2 in human peripheral blood mononuclear cells.

OBJECTIVE: Dimethyl fumarate (DMF) is an anti-inflammatory and antioxidant drug used to treat multiple sclerosis, but its mechanism(s) of action are not fully understood. In central nervous system (CNS) cells, DMF activates nuclear factor E2-related factor 2 (Nrf2), perhaps ameliorating oxidative stress-induced damage. However, it is not known whether DMF also activates Nrf2 in peripheral immune cells, which are known to participate in CNS demyelination. We conducted a single observation study to determine whether DMF can activate Nrf2 in peripheral immune cells in vitro. RESULTS: We performed enzyme-linked immunosorbent assays to measure Nrf2 activation in nuclear extracts of human peripheral blood mononuclear cells treated with DMF at time points from 0 to 6 h, initially determining that DMF did not activate Nrf2, and that the mechanism(s) of action of DMF may thus differ in the periphery compared to the CNS. However, further analyses of our data suggest that high Tmax variability is masking Nrf2 activation in individual donors. Additionally, there may be sub-populations of responders, perhaps related to genetic polymorphisms in Nrf2.

Dimethyl fumarate activates the prostaglandin EP2 receptor and stimulates cAMP signaling in human peripheral blood mononuclear cells.

Dimethyl fumarate (DMF) was recently approved by the FDA for the treatment of relapsing remitting MS. The pathology of MS is a result of both immune dysregulation and oxidative stress induced damage, and DMF is believed to have therapeutic effects on both of these processes. However, the mechanisms of action of DMF are not fully understood. To determine if DMF is able to activate signaling cascades that affect immune dysregulation, we treated human peripheral blood mononuclear cells with DMF. We discovered that DMF stimulates cyclic adenosine monophosphate (cAMP) production after 1 min treatment in vitro. cAMP is a small molecule second messenger that has been shown to modulate immune response. Using pharmacological inhibitors, we determined that adenylyl cyclase mediates DMF induced cAMP production; DMF activated the prostaglandin EP2 receptor to produce cAMP. This response was not due to increased endogenous production of prostaglandin E2 (PGE2), but was enhanced by addition of exogenous PGE2. Furthermore, we determined that the bioactive metabolite of DMF, monomethyl fumarate (MMF), also stimulates cAMP production. These novel findings suggest that DMF may provide protection against MS by inhibiting immune cell function via the cAMP signaling pathway.

Age and immune status of rhesus macaques impact simian varicella virus gene expression in sensory ganglia.

Simian varicella virus (SVV) infection of rhesus macaques (RMs) recapitulates the hallmarks of varicella-zoster virus (VZV) infection of humans, including the establishment of latency within the sensory ganglia. Various factors, including age and immune fitness, influence the outcome of primary VZV infection, as well as reactivation resulting in herpes zoster (HZ). To increase our understanding of the role of lymphocyte subsets in the establishment of viral latency, we analyzed the latent SVV transcriptome in juvenile RMs depleted of CD4 T, CD8 T, or CD20 B lymphocytes during acute infection. We have previously shown that SVV latency in sensory ganglia of nondepleted juvenile RMs is associated with a limited transcriptional profile. In contrast, CD4 depletion during primary infection resulted in the failure to establish a characteristic latent viral transcription profile in sensory ganglia, where we detected 68 out of 69 SVV-encoded open reading frames (ORFs). CD-depleted RMs displayed a latent transcriptional profile that included additional viral transcripts within the core region of the genome not detected in control RMs. The latent transcriptome of CD20-depleted RMs was comparable to the latent transcription in the sensory ganglia of control RMs. Lastly, we investigated the impact of age on the establishment of SVV latency. SVV gene expression was more active in ganglia from two aged RMs than in ganglia from juvenile RMs, with 25 of 69 SVV transcripts detected. Therefore, immune fitness at the time of infection modulates the establishment and/or maintenance of SVV latency.

α-Tocopherol (vitamin E) stimulates cyclic AMP production in human peripheral mononuclear cells and alters immune function.

α-Tocopherol, the most biologically active member of the vitamin E family of fat soluble compounds, exhibits both antioxidant and anti-inflammatory properties. However, its mechanisms of action are not fully understood. Here, we show that, unlike other antioxidants, α-tocopherol stimulates the production of cyclic adenosine monophosphate (cAMP). Inhibitor studies demonstrate that the prostaglandin EP2 and EP4 receptors and adenylyl cyclases mediate the effects of α-tocopherol on cAMP production. Additionally, we show that α-tocopherol attenuates pro-inflammatory cytokine and chemokine production. This study provides novel evidence that α-tocopherol stimulates cAMP signaling, suggesting a mechanism of action for the immunomodulatory effects of vitamin E.

Attenuation of the adaptive immune response in rhesus macaques infected with simian varicella virus lacking open reading frame 61.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Infection of rhesus macaques (RM) with the homologous simian varicella virus (SVV) recapitulates hallmarks of VZV infection. We have shown that an antisense transcript of SVV open reading frame 61 (ORF61), a viral transactivator, was detected most frequently in latently infected RM sensory ganglia. In this study, we compared disease progression, viral replication, immune response, and the establishment of latency following intrabronchial infection with a recombinant SVV lacking ORF61 (SVVΔORF61) to those following infection with wild-type (WT) SVV. Varicella severity and viral latency within sensory ganglia were comparable in RMs infected with SVVΔORF61 and WT SVV. In contrast, viral loads, B and T cell responses, and plasma inflammatory cytokine levels were decreased in RMs infected with SVVΔORF61. To investigate the mechanisms underlying the reduced adaptive immune response, we compared acute SVV gene expression, frequency and proliferation of dendritic cell (DC) subsets, and the expression of innate antiviral genes in bronchoalveolar lavage (BAL) samples. The abundance of SVV transcripts in all kinetic classes was significantly decreased in RMs infected with SVVΔORF61. In addition, we detected a higher frequency and proliferation of plasmacytoid dendritic cells in BAL fluid at 3 days postinfection in RMs infected with SVVΔORF61, which was accompanied by a slight increase in type I interferon gene expression. Taken together, our data suggest that ORF61 plays an important role in orchestrating viral gene expression in vivo and interferes with the host antiviral interferon response.

Improving immunity in the elderly: current and future lessons from nonhuman primate models.

The immune system must overcome daily challenges from pathogens to protect the body from infection. The success of the immune response to infection relies on the ability to sense and evaluate microbial threats and organize their elimination, while limiting damage to host tissues. This delicate balance is achieved through coordinated action of the innate and adaptive arms of the immune system. Aging results in several structural and functional changes in the immune system, often described under the umbrella term "immune senescence". Age-related changes affect both the innate and adaptive arms of the immune system and are believed to result in increased susceptibility and severity of infectious diseases, which is further exacerbated by reduced vaccine efficacy in the elderly. Therefore, multiple strategies to improve immune function in the aged are being investigated. Traditionally, studies on immune senescence are conducted using inbred specific pathogen free (SPF) rodents. This animal model has provided invaluable insight into the mechanisms of aging. However, the limited genetic heterogeneity and the SPF status of this model restrict the successful transfer of immunological discoveries between murine models and the clinical setting. More recently, nonhuman primates (NHPs) have emerged as a leading translational model to investigate immune senescence and to test interventions aimed at delaying/reversing age-related changes in immune function. In this article, we review and summarize advances in immuno-restorative approaches investigated in the NHP model system and discuss where the NHP model can support the development of novel therapeutics.

Simian varicella virus gene expression during acute and latent infection of rhesus macaques.

Varicella zoster virus (VZV) is a neurotropic α-herpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Reactivation of VZV results in herpes zoster and other neurological complications. Our understanding of the VZV transcriptome during acute and latent infection in immune competent individuals remains incomplete. Infection of rhesus macaques with the homologous simian varicella virus (SVV) recapitulates the hallmarks of VZV infection. We therefore characterized the SVV transcriptome by quantitative real-time reverse transcriptase PCR during acute infection in bronchial alveolar lavage (BAL) cells and peripheral blood mononuclear cells, and during latency in sensory ganglia obtained from the same rhesus macaques. During acute infection, all known SVV open reading frames (ORFs) were detected, and the most abundantly expressed ORFs are involved in virus replication and assembly such as the transcriptional activator ORF 63 and the structural proteins ORF 41 and ORF 49. In contrast, latent SVV gene expression is highly restricted. ORF 61, a viral transactivator and latency-associated transcript, is the most prevalent transcript detected in sensory ganglia. We also detected ORFs A, B, 4, 10, 63, 64, 65, 66, and 68 though significantly less frequently than ORF 61. This comprehensive analysis has revealed genes that potentially play a role in the establishment and/or maintenance of SVV latency.

CD4 T cell immunity is critical for the control of simian varicella virus infection in a nonhuman primate model of VZV infection.

Primary infection with varicella zoster virus (VZV) results in varicella (more commonly known as chickenpox) after which VZV establishes latency in sensory ganglia. VZV can reactivate to cause herpes zoster (shingles), a debilitating disease that affects one million individuals in the US alone annually. Current vaccines against varicella (Varivax) and herpes zoster (Zostavax) are not 100% efficacious. Specifically, studies have shown that 1 dose of varivax can lead to breakthrough varicella, albeit rarely, in children and a 2-dose regimen is now recommended. Similarly, although Zostavax results in a 50% reduction in HZ cases, a significant number of recipients remain at risk. To design more efficacious vaccines, we need a better understanding of the immune response to VZV. Clinical observations suggest that T cell immunity plays a more critical role in the protection against VZV primary infection and reactivation. However, no studies to date have directly tested this hypothesis due to the scarcity of animal models that recapitulate the immune response to VZV. We have recently shown that SVV infection of rhesus macaques models the hallmarks of primary VZV infection in children. In this study, we used this model to experimentally determine the role of CD4, CD8 and B cell responses in the resolution of primary SVV infection in unvaccinated animals. Data presented in this manuscript show that while CD20 depletion leads to a significant delay and decrease in the antibody response to SVV, loss of B cells does not alter the severity of varicella or the kinetics/magnitude of the T cell response. Loss of CD8 T cells resulted in slightly higher viral loads and prolonged viremia. In contrast, CD4 depletion led to higher viral loads, prolonged viremia and disseminated varicella. CD4 depleted animals also had delayed and reduced antibody and CD8 T cell responses. These results are similar to clinical observations that children with agammaglobulinemia have uncomplicated varicella whereas children with T cell deficiencies are at increased risk of progressive varicella with significant complications. Moreover, our studies indicate that CD4 T cell responses to SVV play a more critical role than antibody or CD8 T cell responses in the control of primary SVV infection and suggest that one potential mechanism for enhancing the efficacy of VZV vaccines is by eliciting robust CD4 T cell responses.