Recently, 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 HIV positive 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,microgreen flood table 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, higher neurocognitive 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 HIV infected individuals enrolled in the National NeuroAIDS Tissue Consortium cohort for whom CCL2 genotyping and CSF samples were available. The NNTC is a multi-centre 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. All participants had cognitive symptoms of sufficient severity to warrant a HAND diagnosis. 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 .
Genotyping was conducted on a subset of NNTC participants as part of a previously reported study. Peripheral blood mononuclear cells and/or frozen tissue samples were shipped to the University of California Los Angeles Biological Samples Processing Core from the four NNTC sites for DNA extraction.Sample purity was determined via OD 260/280. Extracted DNA was then sent to the UCLA Genotyping Core for genotyping. Prior to genotyping, the samples were checked for concentration by Quant-iT ds DNA Assay kit and for quality by agarose gel. DNA amplification by PCR was performed on 96 and 384-well plates on GeneAmp PCR System 9700 thermal cyclers . Genotypes were determined using the allelic discrimination assay on an Applied Bio-systems 7900 Taqman instrument analysed with SDS2.3 software. Data then underwent error-checking and data-cleaning, including control checks, duplicates checks and checking for Hardy–Weinberg equilibrium. Each genotype was evaluated independently according to a number of quality parameters. Data from cases with genotyping success rate of less than 75% were removed. [Note that brain tissue had poorer genotyping success rate than PBMCs ]. For the CCL2 rs1024611 SNP, haplotype analysis has shown that the rs1024611G polymorphism is associated with allelic expression imbalance of CCL2 and the allele containing -2578G is preferentially transcribed. Within this sample, only 5% of participants were homozygous for the – 2578G allele ; therefore, consistent with previous reports, we combined GA and GG genotypes for statistical analyses to test the hypothesis that carriers of the -2578G allele would express higher levels of CCL2 and greater neurocognitive deficit than noncarriers containing the -2578A allele .Global neurocognitive functioning was determined with a comprehensive battery of neuropsychological measures that included the Trail Making Test , Hopkins Verbal Learning Test, Brief Visuospatial Memory Test, Paced Auditory Serial Addition Test, Wisconsin Card Sorting Test-64 Card Version, Grooved Pegboard, Letter-Number Sequencing, Digit Symbol Test, Controlled Oral Word Association Test and Symbol Search . Validated standard approaches were used in transforming raw scores into standardized T scores and deficit scores. A Global Deficit Score was calculated on the basis of averaging individual test deficit scores.
In line with previous reports,seedling grow rack 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, 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 super family, plays a critical role in mediating B-cell differentiation, activation and survival to generate efficient B-cell responses [30]. Within the CNS, BAFF is expressed by microglia and astrocytes, with recombinant BAFF inducing secretion of inflammatory markers, IL-6 and TNF-a, and IL-10, highlighting BAFF’s contribution to the inflammatory response [31]. 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.