The results of this study suggest that the degree of effectiveness of ARV medications against HIV infection in monocytes, as assessed by in vitro assays in macrophages, is associated with cognitive impairment even among individuals with suppressed plasma HIV RNA. The results may provide insight into a potential mechanism of ongoing brain injury despite meeting standard of care treatment for HIV.
Our study was conceived based on the hypothesis that cells of the monocyte lineage are a protected reservoir for HIV and that these cells are central to the pathogenesis of NCI in the era of HAART. Most of the attention in the literature on reservoirs that support HIV replication has focused on lymphoid cell reservoirs. As a consequence, the role of myeloid cells, and in particular monocytes and macrophages, in viral replication has received far less attention. Although there is much evidence that tissue macrophages support viral replication in vivo
, there is no consensus as to whether macrophages are a viral reservoir in patients on ARV therapy. A recent study from Deleage et al
] showed the presence of HIV in macrophages within seminal vesicles of patients on effective HAART. Similarly, there has been little focus on the extent to which circulating monocytes, the precursor to the tissue macrophage, are infected in patients on or off therapy and again, little consensus as to whether they constitute a viral reservoir in vivo
]. However, recent studies from Spivak et al
] examined the frequency of infected monocytes in elite controllers. Although monocyte infection was undetectable in elite controllers, they reported infected monocytes in a few subjects on HAART. Collectively, these studies implicate monocyte and macrophage in viral persistence and additional studies are warranted in order to fully determine the extent to which myeloid lineage cells support viral persistence in the presence and absence of suppressive therapy.
The potential role of monocytes and macrophages in CNS disease is suggested by human autopsy studies, which have shown that there is continued CNS neuroinflammation with high numbers of macrophages in brain despite potent ARV therapy [30
] and from studies in the simian model, which suggest that these macrophages originate from bone-marrow-derived monocytes that traffic through the bloodstream into the brain [14
]. Studies by our group have further linked NCI to higher levels of HIV DNA within monocytes (CD14+
T-cells) in ARV-naive subjects as well as among subjects with suppressed plasma HIV RNA using typical ARV regimens [16
]. As currently available ARV medications vary in their efficacy in macrophages, we hypothesized that the degree of effectiveness of these ARV medications in blood monocytes and tissue macrophages may relate to the degree of NCI in HIV-infected subjects on ARV therapy.
Our model made use of the in vitro
values of ARVs measured using the ‘acute infection’ macrophage model published by Gavegnano and Schinazi [18
] for correlation with neurocognitive end points. This assay methodology, which makes use of the standardized patient viral isolates M-R5 HIV-1BaL
and M-R5 HIV-1SRA1433
macrophages from a pooled aggregate of donors, and hyperactivation using M-CSF, has typically produced an assay variability of approximately 5% in the laboratory of RFS. Specification of the model is important because EC50
values differ greatly between the ‘acute’ and ‘chronic’ in vitro
macrophage models [19
]. The terminology of ‘acute infection’ and ‘chronic infection’ used in published literature in reference to in vitro
assays in macrophages is unfortunate as it does not equate to acute infection or chronic infection in the clinical sense. In the in vitro
acute infection model, macrophages are incubated with drug prior to exposure to HIV. This model therefore is meant to provide information about how effective the drug is in preventing HIV infection of macrophages. By contrast, in the in vitro
chronic infection model, macrophages are incubated with HIV prior to exposure to drug, providing theoretical information of whether HIV infection can be inhibited or eradicated after cells are already infected. Although both may be important in chronically infected patients, we hypothesized that prevention of monocytes/macrophages may be the central factor; hence our use of the acute infection in vitro
data. It should also be pointed out that most ARVs, as a general rule, are ineffective in the in vitro
chronic infection model, making EC50
s from this chronic infection model impractical as a sole measure of efficacy. It is possible that a formula combining aspects of both the acute and chronic in vitro
model may improve accuracy in estimating clinical efficacy.
The strong negative association between the ME score and the PBMC HIV DNA lends theoretical support that the ARV regimens with higher ME scores might affect cognition at least partially by preventing HIV infection of peripheral blood monocytes. Our study utilized HIV DNA within PBMCs as the only available data and not specifically within monocytes. However, PBMC HIV DNA appears to strongly correlate with HIV DNA values specifically within CD14+
T-cells when assessed in the context of cognitive impairment [17
Our study also suggested a weak association between the CPE score and NCI, but only in the cognitive diagnoses and without pairwise differences between groups. This is not inconsistent with our finding that the ME score is associated with cognition as the two hypotheses are not mutually exclusive. Both hypotheses are based on scientifically sound principles that current ARV therapies do not sufficiently target reservoirs that are important to the CNS. The CPE hypothesis posits that ARV medications must be present in the CSF at levels that exceed the median inhibitory concentration as defined in plasma or in vitro
]. The inference is that these levels are reflective of effectiveness in CNS. The ME hypothesis similarly targets effectiveness of therapy but against monocytes/macrophages whether in the bloodstream or brain as the focus of interest, with the rationale that these cells upon transmigration to the CNS are the primary ongoing source for proinflammatory cytokines and viral replication responsible for synapto-dendritic injury and cognitive consequences [14
]. Since the effect of ARV therapy on monocytes/macrophages in relation to CNS outcomes may also require that drugs adequately penetrate the CNS, it is plausible that the most effective treatments will be those that both minimize HIV intracellular reservoir in monocytes/macrophages and penetrate well into the CNS. It is conceivable that combining the elements of both the ME score and the CPE score into a single score may improve the predictive value above each score independently.
Together, our new data presented here and our past work related to intracellular HIV DNA provide a mechanistic framework for further investigation based on the hypothesis that current ARV therapy is not able to completely prevent HIV infection of monocytes/ macrophages and that this has relevance to NCI. HIV infection of these cells can be hypothesized to lead to an activated phenotype that would support increased monocyte transmigration, increased concentration of HIV infected macrophages in brain, immune activation and synapto-dendritic dysfunction resulting in cognitive compromise [14
]. With persistent use, combinations of ARV medications with higher effectiveness in this reservoir may slowly decrease intracellular burden over time in this cell lineage by preventing new infections prior to leaving bone marrow, while circulating in the blood stream, or among macrophages and microglial cells in CNS tissue.
This study has some important limitations. The study entry criteria of stable ARV therapy ≥6 months was chosen based on the expected duration of time needed to result in maximal repression of plasma HIV RNA. However, this inclusion criteria, together with the lack of EC50 (acute infection) values for atazanavir and lopinavir resulted in exclusion of approximately half of the original HAHC population, which may have introduced bias into the study. Furthermore, the EC50 data comes from in vitro assays performed in macrophages and not blood monocytes and the ME score formula does not account for the unique pharmacodynamics/ kinetics of each drug. The HAHC study was conducted between 2001 and 2006, at a time when newer ARV medications such as etravirine, darunavir, maraviroc and raltegravir were not commercially available. Future studies will need to validate whether the efficacy of the ME score in predicting cognitive status continues to apply in an HIV-infected population on more current medications.
The results of this study are intriguing but require validation before its premises can be accepted as valid. If validated, however, our findings may have clinical significance for strategies on how to address the neurological complications of HIV. These findings may also have relevance to attempts to eradicate HIV. Recent review articles focus on clearance of HIV from the latent CD4+
T-cell infected reservoir; yet equal attention may be required for the monocyte/macrophage reservoir [33
]. As these cells serve as primary antigen presenting cells for CD4+
T-lymphocytes and a primary source of new infection for these cells, control of monocyte/ macrophage infection may be a crucial part of reservoir eradication. Prospective studies designed to determine if ARV therapy with a higher ME score will have a superior effect on cognition may need to consider the extended half-life of activated cells of the monocyte lineage, estimated to be from months to years [35