In this study we examined both monocyte and T cell populations to understand the impact of HIV/HCV coinfection on peripheral immune cells and how that may relate to cognition. We selected CD14 monocytes for gene expression analysis based on their immune response characteristics, including sensitivity to LPS, and because they could be isolated rapidly at high purity from whole blood thereby preserving their in vivo
phenotype. For these analyses, we included monocyte gene expression data obtained from HIV-infected individuals with viremia or undetectable viral loads that was previously published 
. Gene expression analysis of monocytes from HCV and HIVUD
subjects revealed distinct expression profiles with significant induction of monocyte genes in HCV-infected subjects. In contrast, monocyte expression profiles in HIVUD
subjects were similar to healthy controls. Furthermore, monocyte genes upregulated in HCV subjects were comparably expressed in HIV-suppressed coinfected subjects, while HIV-related genes in the HIV-suppressed coinfected were similar to those in HIV subjects but not induced in HIVUD
subjects. A possible explanation for the activated profile in HIV-suppressed coinfected subjects is that HIV primes monocytes to be hypersensitive to agonists that activate the innate immune response 
. While HCV in chronically infected subjects did not elicit an HIV expression profile, HCV in the context of HIV priming may trigger monocyte activation. Monocytes harvested from HIV-infected individuals and analyzed for gene expression by us and others have identified a type 1 IFN response where the level of activation was dependent on viral load 
. However, HIV viral load is unlikely driving the IFN response in HIV-suppressed coinfected subjects since HIV was undetectable. Our finding is consistent with an earlier report of an aberrant type1 IFN response in HIV-suppressed coinfected individuals also identifying upregulation of IFI27, IFI44 and MX2 in PBMCs 
. Interestingly, immune activation was reduced following effective peg-IFN/ribavirin treatment in individuals who achieved HCV sustained viral response 
. These results are consistent with monocyte activation being triggered by factors other than HIV viral load and implicate HCV infection through, as yet, an undefined mechanism triggering immune activation in HIV-suppressed coinfected subjects.
In this cohort, we found that coinfection was associated with significantly greater cognitive dysfunction, greater than that in either HCV or HIV monoinfected subjects. Numerous studies have evaluated the neuropsychological status of HIV-suppressed coinfected individuals and the literature is divided between studies which did not find a difference between HIV-suppressed coinfected and HCV monoinfected patients 
and those which found worse cognitive performance in the HIV-suppressed coinfected 
. Even when impairment was identified, it is often judged to be mild, which implies that subtle differences in cohort recruitment as well as the choice of particular neuropsychological tests may have a considerable impact on the cognitive characterization of HIV-suppressed coinfected subjects. In this cross-sectional study, we attempted to minimize confounding factors by recruiting HIV-suppressed coinfected subjects with undetectable HIV viral loads who were receiving stable medical care, had no significant liver disease and were infected with HCV genotype 1 with no current drug or alcohol abuse 
. Subjects were assessed for neuropsychological deficits in seven domains thereby increasing sensitivity with resulting T scores converted into GDS. Using Spearman rank coefficient analysis, we identified eight genes, IF127, RSAD2, MX1, ETV7, SIGLEC1, LGALS3BP, C1QC and HES4 from HIV-suppressed coinfected subjects that correlated with GDS thereby linking an expression profile with poorer cognition. Seven were type 1 IFN-related genes while HES4 was upregulated in both HCV and HIV monoinfection and all of the genes were expressed at levels above that found in the HIVUD
. The Hairy/Enhancer of Split 1 (HES1) gene product is a transcriptional repressor implicated in the control of proliferating neural stem cells 
which itself is regulated by notch signaling 
. While HES4 was not induced in monocytes treated with IFNα 
, it was upregulated 7-fold in PBMCs treated with immune complexes that cause inflammation in systemic lupus erythematosus 
. Four of the genes, IFI27, ETV7, SIGLEC1 and LGALS3BP were previously identified in activated monocytes isolated from HIV subjects with viremia where their expression correlated with lower brain metabolite N-acetylaspartate levels in frontal white matter as determined by 1
H magnetic resonance spectroscopy 
. Together these observations point to a relationship connecting coinfection and monocyte activation that impacts cognitive function perhaps by altering brain metabolite levels.
Lipopolysaccharide is a potent proinflammatory agent possibly linked to cognitive impairment. During HIV infection, the gastrointestinal barrier is compromised due to selective loss of CD4 T cells with subsequent bacterial translocation and elevated endotoxin levels in the blood 
. Plasma LPS is elevated in HIV-infected subjects compared to healthy controls 
and was found to correlate with HIV-associated dementia independent of viral load 
. In coinfection, impaired LPS detoxification due to liver damage could contribute to high immune activation levels, which is supported by evidence that microbial translocation is associated with cirrhosis 
. We examined plasma LPS levels in our cohort and found significantly higher LPS in the HIV-suppressed coinfected population compared to healthy controls. We also tested for a correlation between plasma LPS concentration and GDS in the HIV-suppressed coinfected and found no relationship. Based on these observations, LPS does not appear to be a factor causing cognitive impairment in HIV-suppressed coinfected individuals.
We examined the T cell compartment to determine if coinfection was related to an activated phenotype. HIV drives persistent immune activation typified by a higher percentage of T cells expressing activation markers 
. The most effective correlate of HIV disease progression is upregulation of CD38 on CD8 T cells 
with one of the consequences being activation-induced apoptosis in CD4 and CD8 T cells 
. While effective ART in HIV-infected subjects dramatically lowers activation by suppressing HIV viral replication and allowing partial reconstitution of immune function, low-level persistent activation remains 
. Incomplete restoration of normal CD38 expression in HIVUD
subjects was evident in our subjects as well, particularly in the CD8 cells, which expressed significantly elevated CD38 compared to controls and HCV monoinfected subjects. In our cohort, HLADR was the most effective marker for differentiating coinfection in both CD4 and CD8 subsets demonstrating significantly higher expression than either HCV or HIV monoinfection, which extended to the CD38/HLADR population as well. Others who have investigated these markers in HIV-suppressed coinfected subjects have likewise identified activation in the T cell compartment but with somewhat different results. Kovacs et al. found elevated CD38/HLADR in CD8 but not in the CD4 subset 
while Gonzalez et al., examined only CD38 expression which was increased in both CD4 and CD8 subsets in HIV-suppressed coinfected compared to HCV and HIV monoinfection 
. It is important to consider this activation in context of controlled HIV viral load, which in our cohort was undetectable. This suggests an alternative mechanism for peripheral activation in coinfection since viral suppression of HIV is normally sufficient to lower T cell activation markers. In addition, T cell activation did not correlate with cognitive impairment in HIV-suppressed coinfected subjects, which was contrary to expectation. This suggests that monocyte activation in coinfection is not intrinsically associated with T cell activation.
Irrefutable evidence that coinfection increases the risk of cognitive impairment remains elusive. Published reports that detect impairment are counterbalanced by studies that fail to find significant association with coinfection. Considered separately, there is no question of HIV’s role in neurodegeneration and cognition impairment particularly when viral loads are not suppressed. With effective ART, neuropathogenesis has been reduced and cognition improved and yet HIV-associated neurocognitive disorders remain 
. Findings of diminished cognition are equivocal regarding HCV, while more compelling in coinfection. We are the first to report a monocyte activation profile that correlates with cognitive impairment in the HIV-suppressed coinfected population. This implies a risk of impairment even when HIV viral loads are effectively suppressed. Recent improvements in HCV therapy include the addition of highly effective HCV protease inhibitors to the standard peg-IFN/ribavirin therapy 
. This is expected to increase the number of coinfected individuals who are treated and ultimately the number who achieve a sustained viral response (SVR). By effectively suppressing HCV, we anticipate that monocyte activation will decrease with subsequent cognitive improvement in those who previously exhibited a type 1 monocyte expression profile. This conjecture needs to be tested using a longitudinal treatment study that could assess immune activation and cognitive impairment in individuals who maintain SVR.