Genomics Links Inflammation With Neurocognitive Impairment in Children Living With Human Immunodeficiency Virus Type-1

Abstract

Background: We identified host single-nucleotide variants (SNVs) associated with neurocognitive impairment (NCI) in perinatally HIV-infected (PHIV) children.

Methods: Whole-exome sequencing (WES) was performed on 217 PHIV with cognitive score for age (CSA) < 70 and 247 CSA ≥ 70 (discovery cohort [DC]). SNVs identified in DC were evaluated in 2 validation cohorts (VC). Logistic regression was used to estimate adjusted odds ratios (ORs) for NCI. A human microglia NLRP3 inflammasome assay characterized the role of identified genes.

Results: Twenty-nine SNVs in 24 genes reaching P ≤ .002 and OR ≥ 1.5 comparing CSA < 70 to CSA ≥ 70 were identified in the DC, of which 3 SNVs were identified in VCs for further study. Combining the 3 cohorts, SNV in CCRL2 (rs3204849) was associated with decreased odds of NCI (P < .0001); RETREG1/FAM134B (rs61733811) and YWHAH (rs73884247) were associated with increased risk of NCI (P < .0001 and P < .001, respectively). Knockdown of CCRL2 led to decreased microglial release of IL-1β following exposure to ssRNA40 while knockdown of RETREG1 and YWHAH resulted in increased IL-1β release.

Conclusions: Using WES and 2 VCs, and gene silencing of microglia we identified 3 genetic variants associated with NCI and inflammation in HIV-infected children.

Keywords: 14-3-3η protein; CCRL2; HIV; HIV perinatal infection; RETREG1; YWHAH; genome-wide exome sequencing; neurocognitive impairment.

Figure 1.

Combined and cohort-specific ORs by SNVs associated with neurocognitive impairment of children. Gray lines indicate 95% confidence intervals and larger box size indicates more precision. Abbreviations: DC, discovery cohort; OR, odds ratio; SNV, single nucleotide variant; VC, validation cohort.

Figure 2.

Knockdown of CCRL2 in human microglial cells results in reduced inflammatory response to HIV ssRNA40 and increased autophagy levels. A, Left, Nonspecific control siRNA (a nontargeting DsiRNA that does not interact with any sequence in human genome; siControl) and siCCRL2-transfected human microglia were assessed for CCRL2 mRNA expression at 48 hours by qPCR. Both control and CCRL2-depleted microglial cultures were incubated with LyoVec (vehicle) or ssRNA40 (5 µg/mL) for an additional 24 hours. Culture supernatants were collected for ELISA, and cells were collected and proteins extracted for immunoblotting. Center, densitometry quantification values were collected for proIL-1β immunoblots after ACTB normalization (see Supplementary Figure1B for representative immunoblots) and plotted based on 4 different donors as means ± SEM. Right, quantitative measurement of IL-1β in the culture supernatants by ELISA (n = 6); median is plotted as horizontal line. B, Left, representative immunoblots for LC3B lipidation (LC3B-II) and SQSTM1. Center and right, densitometry quantification values were collected after ACTB normalization (see Supplementary Methods for details) and plotted based on 6 different donors as means ± SEM. *P < .05, **P < .005, ***P < .001. Abbreviations: ACTB, β-actin; DsiRNA, Dicer-substrate small interfering RNA; ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; IL-1β, interleukin-1β; LC3B, light chain 3B; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; SQSTM1, sequestosome 1.

Figure 3.

Knockdown of RETREG1 (FAM134B) and YWHAH (14-3-3η) in human microglial cells results in enhanced inflammatory response to HIV ssRNA40 and inhibition of autophagic flux. A, Left, nonspecific control siRNA (a nontargeting DsiRNA that does not interact with any sequence in human genome; siControl) and siRETREG1 transfected human microglia were assessed for RETREG1 mRNA expression at 48 hours by qPCR. Both control and RETREG1-depleted microglial cells were incubated in culture with LyoVec (vehicle) or ssRNA40 (5 µg/mL) for an additional 24 hours. Culture supernatants were collected, microglia were harvested, proteins extracted and assessed by immunoblotting. Center, densitometry quantification values were collected for proIL-1β immunoblots after ACTB normalization (see Supplementary Figure 1C for representative immunoblots and Supplementary Methods for details) and plotted based on 4 different donors as means ± SEM. Right, quantitative measurement of IL-1β in the culture supernatants by ELISA (n = 6); median is plotted as horizontal line. B, Left, representative immunoblots for LC3B lipidation (LC3B-II) and SQSTM1 degradation assessment. Center and right, densitometry quantification values were collected after ACTB normalization (see Supplementary Methods for details) and plotted based on 6 different donors as means ± SEM. C, Left, nonspecific control siRNA (a nontargeting DsiRNA that does not interact with any sequence in human genome; siControl) and YWHAH siRNA (siYWHAH) transfected human microglia were collected at 48 hours for YWHAH mRNA expression analysis by qPCR. Both control and YWHAH-depleted cells were incubated with LyoVec (vehicle) or ssRNA40 (5 µg/mL) for an additional 24 hours. Culture supernatants were collected for ELISA, cells were collected, lysed, and lysates were used for immunoblotting. Center, densitometry quantification values were collected for proIL-1β immunoblots after ACTB normalization (see Supplementary Figure 1D for representative immunoblots and Supplementary Methods for details) and plotted based on 4 different donors as means ± SEM. Right, quantitative measurement of IL-1β in the culture supernatants by ELISA (n = 6); median is plotted as horizontal line. D, Left, representative immunoblots for LC3B lipidation (LC3B-II) and SQSTM1 degradation assessment. Center and right, densitometric quantification values were collected after ACTB normalization (see Supplementary Methods for details) and plotted based on 6 different donors as means ± SEM. *P < .05, **P < .005, ***P < .001. Abbreviations: ACTB, β-actin; DsiRNA, Dicer-substrate small interfering RNA; ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; IL-1β, interleukin-1β; LC3B, light chain 3 B; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; SQSTM1, sequestosome 1.