We, and others, have previously demonstrated that TNFα produced from HIV-1 infected or activated macrophage and microglia in the CNS is a major player in HIV-1-induced neuroinflammation, that eventually leads to neuron damage and cognitive impairment 
. Both neuroinflammation and monocyte/macrophage infiltration into the CNS are critical factors in the development of HAND, despite effective control of HIV-1 levels with HAART. Antiretroviral drugs target and effectively control viral replication, but once proviral DNA has integrated into the host chromosome, the production of early viral proteins such as Tat, a known neurotoxin, is not affected by these antiretrovirals 
. It is also important to consider the limited CNS penetration and neurotoxicities of many antiretroviral drugs themselves 
. For these reasons, mechanisms to inhibit macrophage and microglial activation and neuroinflammation deserve further investigation in the pursuit of adjunctive therapies designed to protect CNS cells from damage caused by HIV-1. A myriad of possible adjunctive treatment compounds have been tested in vitro
. Compounds targeting cellular signaling pathways including glycogen synthase kinase-3 beta (GSK3β) and mixed lineage kinase-3 (MLK3), as well as compounds targeting pathological outcomes of HIV-1 infection in the CNS including excitotoxicity, oxidative stress, and inflammation showed initial promise in vitro
, however, only a handful of these treatments have moved into clinical trials. To date, only memantine, an NMDAR blocker, has preliminarily shown the ability to protect patients from HIV-1-induced neurocognitive damage 
. The persistence of HAND despite effective HAART treatment, together with the lack of effective available adjunctive therapies for HAND, highlight the need for studies investigating novel adjunctive therapies.
In addition to ibudilast, other PDE inhibitors have been considered as anti-inflammatory agents in the context of HIV-1 infection. For example, initial in vitro
experiments with pentoxifylline, a non-selective PDE inhibitor, showed that this drug inhibited microglial cell activation and the production of pro-inflammatory cytokines 
. Also, in peripheral blood mononuclear cells (PBMCs) from HIV-1 positive, pentoxifylline-treated patients, there was lower LPS-induced TNFα release as compared to cells from HIV-1 positive, non-treated patients 
. However, it was subsequently shown that the maximum tolerated dose of pentoxifylline in patients did not produce plasma concentrations high enough to match concentrations necessary to achieve the anti-inflammatory effects seen in vitro 
. Additionally rolipram, a specific inhibitor of PDE 4, has been shown to suppress cytokine production and to inhibit HIV-1 replication in T-cells 
. The low plasma concentrations of pentoxifylline, together with the gastrointestinal side effects associated with the use of PDE inhibitors, has limited further investigation of several PDE inhibitors for use in the context of HIV-1 infection.
NF-κB signaling plays an important role in the production of TNFα. As such, it has been shown that pentoxifylline inhibits NF-κB as a mechanism for its inhibition of TNFα 
. Also, increased cAMP levels have been shown to inhibit NF-κB transcriptional activity 
. Considering this, we chose to investigate the effect of ibudilast on NF-κB as a potential mechanism for its inhibition of TNFα. As predicted, we did see a significant inhibition of Tat-induced NF-κB activation by ibudilast. Interestingly, we did not see a reversal of Tat-mediated IκBα degradation with ibudilast pre-treatment. This lack of IκBα stabilization by ibudilast suggests that the NF-κB inhibition by ibudilast occurs after RelA nuclear translocation, which is mediated by IκBα degradation. The transcriptional activity of RelA is partially controlled by multiple post-translational modifications, including phosphorylation, ubiquitination, and acetylation 
. However, we did not see a reduction in Tat-induced RelA phosphorylation with ibudilast pre-treatment. Other possibilities for NF-κB inhibition in the nucleus include additional post-translational modifications, such as acetylation or ubiquitination, dimer exchange, or interactions with nuclear proteins such as the transcriptional coactivator and acetyltransferase, p300. Given the complexity of the regulation of NF-κB activity, it is not surprising that ibudilast's inhibition of NF-κB signaling appears to involve a post-translational modification of NF-κB, following normal mobilization of these molecules into the nucleus. Ibudilast could also potentially be inhibiting the DNA binding activity of NF-κB, as has been reported with the type 3 PDE inhibitor, cilostazol 
. The precise mechanism of ibudilast's action on NF-κB transcriptional activity is currently under investigation in our laboratory.
In addition, there are hundreds of NF-κB inhibitors, all of which have greatly contributed to NF-κB research, but have limited clinical applications because of considerable toxicity to healthy cells, as NF-κB signaling is intricately involved in multiple cellular processes 
. Considering the multiple functions of NF-κB signaling, specifically in the CNS 
, drugs such as ibudilast, with only partial inhibition of NF-κB in the context of HIV-1 in the CNS, could be beneficial.
Based on ibudilast's history of use in patients, this drug deserves further study as an anti-inflammatory agent in the context of HIV-1-induced neuroinflammation. Recent studies have shown that combinations of PDE inhibitors can synergistically suppress TNFα production by microglia at much lower concentrations than any single PDE inhibitor alone 
. This could circumvent the problem of low patient plasma concentrations encountered with pentoxifylline treatment. It is also important to emphasize the tolerability of ibudilast in patients, as only mild gastrointestinal side effects, such as nausea and diarrhea, have been reported 
. Indeed, ibudilast has been used for decades to treat bronchial asthma in Japan, and is being tested in clinical trials as a treatment for MS, opioid withdrawal, and neuropathic pain. Additionally, ibudilast has been shown to effectively cross the blood brain barrier and has been measured in rat brain tissue at concentrations close to plasma levels 
Considering ibudilast's potential ablity to inhibit or resolve CNS inflammation, there could be additional benefits such as limiting viral spread by reducing the inflammation-induced influx of monocytes into the CNS. In addition, TNFα and other related cytokines have wide-ranging neuromodulatory and even neuroprotective functions 
. As such, ibudilast's partial inhibition of Tat-induced TNFα production could be particularly useful when considering this drug as an adjunctive therapy for HAND. Complete inhibition of TNFα production is undesirable, as this could negatively impact TNFα's ability to perform efficient host defense. In this respect, it has been shown that TNFα blockade can cause reactivation of tuberculosis (TB) in animal models 
, and it has also been suggested that inhibition of TNFα in patients with chronic hepatitis B infection (HBV) could worsen the disease, since TNFα is involved in viral clearance 
. Furthermore, inhibition of TNFα in the context of other neuroinflammatory disorders such as MS and Alzheimer's disease (AD) has very recently been shown to be associated with progressive multifocal leukoencephalopathy (PML) and demyelination 
. Taken together, these considerations again emphasize that ibudilast's partial inhibition of TNFα could be beneficial, rather than detrimental. In summary, our findings shed light on the mechanism of ibudilast's inhibition of Tat-induced TNFα production in microglial cells and may implicate ibudilast as a potential adjunctive therapy for the management of HAND.