Despite the success of antiretroviral therapy, approximately 20–50% of HIV-infected individuals have HIVassociated neurocognitive disorder. HIV infection and stimulation of monocytes and lymphocytes promotes trafficking into the central nervous system, triggering a neuroinflammatory response. Within the CNS, inflammation leads to activation of microglia, the resident immune cells in the brain, which induces chemokines and cytokines that drive a chemotactic gradient along the blood -brain barrier and allows further infiltration of infected and uninfected peripheral immune cells. Chemokines and cytokines function as immunomodulatory proteins that influence HIV neuropathogenesis with both positive and negative effects that may contribute to the ongoing prevalence of HAND. The chemokine C-C motif ligand 2 , alternatively known as monocyte chemoattractant protein-1 , is a b-chemokine that is expressed during inflammation and that, upon activation of its receptor , can induce chemotaxis of monocytes to inflammatory sites generated by injury and infectious events. CCL2 is expressed by monocytes, macrophages, dendritic cells, neurons, astrocytes, microglia and endothelial cells, while CCR2 is expressed by monocytes, microglia, astrocytes, epithelial cells, activated T cells and dendritic cells. CCL2 has been identified as the most potent activator of macrophages in comparison to other monocyte chemoattractants, including RANTES, macrophage inflammatory protein-1a , MIP- 1 b, MCP-2 b and MCP-3. CCL2 levels in the brain and cerebrospinal fluid are elevated in HIV patients with encephalitis, AIDS patients with cytomegalovirus, AIDS dementia and HIV-positive patients with cerebral inflammation. Recently,pruning cannabis peripheral blood monocyte expression of CCR2 has been shown to predict HAND in combination ART – era HIV cohorts. Elevated levels of CCL2 expression have also been observed in non HIV-positive samples.
In patients with mild cognitive impairment and Alzheimer’s disease, higher levels of CSF CCL2 correlated with lower cognitive scores. The presence of elevated CCL2 in HIV-positive patients with neuroinflammation, predictive power of monocyte CCR2 for HAND and elevations of CCL2 in non HIVpositive patients with neurocognitive deficits suggests that CCL2 may have a critical role in the neuropathogenesis of HAND and other noninfectious dementing disorders. A number of studies have examined genetic variation in the CCL2 gene and identified single nucleotide polymorphisms to be associated with HIV-disease progression and neurocognitive functioning over time. Individuals with an A to G polymorphism in the CCL2 enhancer region, annotated as rs1024611 , have higher CCL2 levels in serum, plasma and CSF than individuals without the A to G polymorphism. Increased CCL2 expression from the – 2578G allele has also been investigated in pathologic conditions and was found to be associated with higher incidences of tuberculosis, breast cancer and atherosclerosis, suggesting that the SNP is involved in chronic inflammatory conditions. Among HIV-infected individuals, homozygosity for the -2578G allele was associated with accelerated disease progression, enhanced leukocyte recruitment to tissues and a 4.5-fold risk for HIV-associated dementia. In a study examining the -2578G allele in a cognitively impaired population, elderly patients with senile dementia due to Alzheimer’s disease, CCL2 serum levels were significantly higher in patients who carried at least one G allele, whereas the highest levels of CCL2 were present in patients carrying two G alleles. The -2578G allele has also been reported to be associated with diminished performance in working memory over time in HIV infected individuals. The HIV-positive group who did not carry the -2578G allele improved at a faster rate in working memory than the HIV-positive group who carried the -2578G allele, but not faster than the HIVnegative groups. However, direct associations between the CCL2 rs1024611 SNP and HIV-disease progression have not been consistent across studies , suggesting that there may be intermediate mechanisms that mediate the association between CCL2 genotype, host immune responses and neurocognitive outcomes.
Although CCL2 expression in both plasma and serum has been linked to neurocognitive impairment, the purpose of the current study was to elucidate interrelationships between CCL2 genotype at the rs1024611 SNP, CCL2 levels in CSF, expression of other neuroinflammatory markers in the CSF. In addition, we considered plasma viral load, CD4þ cell count and neurocognitive performance in our analysis of HIV-infected individuals. We hypothesized that HIV-positive carriers of the CCL2 -2578G allele would exhibit high levels of CCL2 expression in CSF, and that elevated levels of CCL2 would be associated with higher concentrations of other proinflammatory markers in CSF, higherneurocognitive deficit scores, higher HIV viral load and a lower CD4þ T-cell count in blood plasma. We also hypothesized that accounting for CSF levels of CCL2 would explicate the relationship between CCL2 genotype and cognition.The cohort that was examined consisted of 145 HIVinfected individuals enrolled in the National NeuroAIDS Tissue Consortium cohort for whom CCL2 genotyping and CSF samples were available. The NNTC is a multicentre consortium engaged in a longitudinal study of adults with HIV/AIDS. The four participating clinical centres in the United States were The National Neurological AIDS Bank located in Los Angeles, California, USA; the Texas NeuroAIDS Research Center located in Galveston, Texas, USA; the Manhattan HIV Brain Bank located in New York, New York, USA; and the California NeuroAIDS Tissue Network located in San Diego, California, USA. Participants are administered a comprehensive battery of psychometric measurements that include tests of neuropsychological function and self-report instruments that estimate past and current substance and psychiatric illness. Neurological examinations, lumbar puncture for CSF collection and laboratory tests were conducted for plasma HIV viral load and plasma CD4þ lymphocyte count. The following were the inclusion criteria in the current study: at least 18 years of age, fluent in the English language, at least sixth grade education, able to provide informed consent.
Exclusion criteria were as follows: no history of CNS opportunistic infections , no history of traumatic brain injury, no history of learning disability or other developmental disorders and no other major neurologic syndromes .In line with previous reports, the results of this study showed that carriers of the -2578G allele had significantly higher levels of CCL2 in CSF. In addition, higher CCL2 expression was correlated with neurocognitive deficit score, higher levels of other proinflammatory markers in CSF, higher plasma viral load and lower CD4þ lymphocyte counts. We did not observe a significant interaction between CCL2 genotype at rs1024611 and neurocognitive deficit score, suggesting that carrying the -2578G allele alone does not appear to effect cognition. Instead, the findings suggest that increased expression of CCL2, modulated by CCL2 genotype, influences neurocognitive test performance. As expected, there was a strong correlation between CCL2 genotype and CCL2 expression, which led to the investigation of whether CSF CCL2 expression was acting as a moderating variable to the effects of CCL2 genotype on cognition. This suggests that the -2578G genotype results in a more reactive immune response, and increased viral load results in higher concentrations of CCL2 than normal, resulting in neurocognitive dysfunction. After controlling for CCL2 expression, the association between genotype and cognition emerged, indicating that CCL2 genotype has an effect on cognition, which may be moderated by CCL2 expression. Using plasma viral load to further probe the relationship between CCL2 genotype on cognition in the context of HIV infection,drying room we found that in the presence of high viral load, the CCL2 -2578G allele was associated with greater CCL2 expression and neurocognitive deficit. Although plasma viral load was used as a surrogate for CSF viral load , these results suggest that as HIV infection persists, carrying the – 2578G allele will lead to worse cognitive outcomes . The results also suggest that carrying the CCL2 -2578G allele and thereby expressing higher levels of CCL2 may contribute to or support a pro-inflammatory state in the CNS. We found that CSF CCL2 was associated with increased sCD14, sIL-6Ra, IL-2, IL-6, BAFF and sTNFR2. However, we are unable to determine whether increased CCL2 expression is a consequence of an already established pro-inflammatory state or if induction of CCL2 drives the pro-inflammatory immune response. Interestingly, in addition to CCL2, we found that BAFF and sTNFR2 correlated with cognitive performance; however, CCL2 was the only marker that was associated with CCL2 genotype. These results suggest that BAFF and sTNFR2 may also play an important role in neuroinflammation and cognitive impairment among HIV-infected individuals. B-cell activating factor , a cytokine that is a member of the TNF superfamily, plays a critical role in mediating B-cell differentiation, activation and survival to generate efficient B-cell responses.
Within the CNS, BAFF is expressed by microglia and astrocytes, with recombinant BAFF inducing secretion ofinflammatory markers, IL-6 and TNF-a, and IL-10, highlighting BAFF’s contribution to the inflammatory response. Consistent with our results, elevated levels of BAFF in CSF from patients with inflammatory neurological diseases, including HIV, have been reported to be significantly higher than in patients with nonin- flammatory neurological diseases. These findings highlight the importance of controlled BAFF expression for an efficient B-cell response, which is a contributing factor in HIV disease progression. Increased CSF BAFF may be indicative of neuroinflammation and may be important in the persistence of HIV within the CNS. TNFR2 is a receptor for TNF-a, a key regulatory cytokine in the inflammatory response, and upon binding, induces a signalling cascade to promote cell survival. TNFR2 is expressed by lymphocytes , microglia, oligodendrocytes, astrocytes, endothelial cells, myocytes, thymocytes and mesenchymal stem cells. Soluble TNFR2 can be generated via shedding from the cell surface and may act as a scavenger in a protective capacity by sequestering TNF-a to reduce TNF mediated inflammation. TNFR2 signalling in neurologic disorders and cognitive impairment has been examined and increased levels of sTNFR2 have been reported in CSF and plasma from patients diagnosed with bipolar disorder, mild cognitive impairment and AD compared with healthy controls. Increased staining for TNFRs has also been demonstrated in brains of individuals with HIV encephalitis and other opportunistic infections, and one report has described an association of plasma sTNFR2 with HIV-associated cognitive abnormalities. These results suggest that increased sTNFR2 expression in CSF may serve as a marker of the neuroinflammatory response to HIV and may play an important role in HIV neuropathogenesis and neurocognitive impairment. Our results indicate that individuals carrying the CCL2 – 2578G allele expressed higher levels of CCL2 in CSF and correlational analyses demonstrated that increased CCL2 was associated with increased pro-inflammatory markers, including sCD14, sIL-6Ra, IL-2, IL-6, BAFF and sTNFR2, in addition to greater cognitive deficit. These results are in line with previous studies reporting that increased levels of CCL2 are associated with a faster rate of cognitive decline. Furthermore, the CCL2- CCR2 axis was recently reported to be a critical signalling pathway in HAND, with CCR2 on CD14þCD16þ monocytes serving as a peripheral biomarker for HAND . Overproduction of cytokines in the CNS may allow for HIV-infected cells to persist in the brain despite antiretroviral therapy treatment. As stated previously, CCL2 expression was also correlated with plasma viral load. This is of particular clinical importance because the failure to adequately suppress viral replication may result in repeated BBB insults that further contribute to peripheral immune cell migration into the CNS. This is an area that requires further investigation and has the potential to inform therapeutic interventions. Owing to the cross-sectional nature of the current study, we cannot determine whether the expression of CCL2 is a precursor, consequence or simply correlative to cognitive status. It is possible that elevations in CCL2 expression in CSF may signal other inflammatory processes or genetic influences that were not evaluated in the current study. For instance, HIV-1 Tat protein is produced in infected astrocytes and may be secreted and taken up by neighbouring cells, and has been identified as a potential factor in the pathophysiology of HAND. Expression of the CCL2 gene has been shown to be directly transactivated by HIV-1 Tat protein in human astrocytes. Although we cannot determine from this study whether the CCL2 gene is solely driving the CCL2 gradient or whether the presence of infected cells or HIV- 1 Tat are also contributing to CCL2 expression, the results suggest a link between CCL2 genotype and cognition that warrants further study. Overall, these results underscore the importance of examining intermediate phenotypes as modulating factors that may link host genotype to cognitive outcomes in HIV.Treatment-seeking individuals with an alcohol use disorder exhibit a range of neurocognitive and inhibitory control deficits.