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. 2022 Nov 29;119(48):e2210584119.
doi: 10.1073/pnas.2210584119. Epub 2022 Nov 21.

Elevation of cell-associated HIV-1 transcripts in CSF CD4+ T cells, despite effective antiretroviral therapy, is linked to brain injury

Kazuo Suzuki  1   2 , John Zaunders  1   2 , Thomas M Gates  3   4   5   6 , Angelique Levert  1 , Shannen Butterly  1 , Zhixin Liu  7 , Takaomi Ishida  8 , Sarah Palmer  9 , Caroline D Rae  10   11 , Lauriane Jugé  10   11 , Lucette A Cysique  3   4   5   6 , Bruce J Brew  2   3   4   5   11   12
Affiliations

Elevation of cell-associated HIV-1 transcripts in CSF CD4+ T cells, despite effective antiretroviral therapy, is linked to brain injury

Kazuo Suzuki et al. Proc Natl Acad Sci U S A. .

Abstract

Antiretroviral therapy (ART) can attain prolonged undetectable HIV-1 in plasma and cerebrospinal fluid (CSF), but brain injury remains prevalent in people living with HIV-1 infection (PLHIV). We investigated cell-associated (CA)-HIV-1 RNA transcripts in cells in CSF and blood, using the highly sensitive Double-R assay, together with proton Magnetic Resonance Spectroscopy (1H MRS) of major brain metabolites, in sixteen PLHIV. 14/16 CSF cell samples had quantifiable CA-HIV-1 RNA, at levels significantly higher than in their PBMCs (median 9,266 vs 185 copies /106 CD4+ T-cells; p<0.0001). In individual PLHIV, higher levels of HIV-1 transcripts in CSF cells were associated with greater brain injury in the frontal white matter (Std β=-0.73; p=0.007) and posterior cingulate (Std β=-0.61; p=0.03). 18-colour flow cytometry revealed that the CSF cells were 91% memory T-cells, equally CD4+ and CD8+ T-cells, but fewer B cells (0.4 %), and monocytes (3.1%). CXCR3+CD49d+integrin β7-, CCR5+CD4+ T-cells were highly enriched in CSF, compared with PBMC (p <0.001). However, CA-HIV-1 RNA could not be detected in 10/16 preparations of highly purified monocytes from PBMC, and was extremely low in the other six. Our data show that elevated HIV-1 transcripts in CSF cells were associated with brain injury, despite suppressive ART. The cellular source is most likely memory CD4+ T cells from blood, rather than trafficking monocytes. Future research should focus on inhibitors of this transcription to reduce local production of potentially neurotoxic and inflammatory viral products.

Keywords: CD4+T cells; brain injury; cerebrospinal fluid; intracellular HIV-1 RNA-transcripts; neuropathogenesis.

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Conflict of interest statement

The authors declare a competing interest. Kazuo Suzuki is the original inventor under WO2018/045425 (PTC/AU2017/050974) patent, titled “Methods of detecting Lentivirus” of HIV-1 detection targeting “R” region. All other authors report no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
Comparison of CSF and PBMC cell numbers and HIV-1 CA-RNA and DNA levels. (A) Range of recovered CSF cell counts for different CD45+ cells obtained in the pellets, including CD3+CD4+ and CD3+CD8+ T lymphocytes, CD14+ monocytes, CD19+ B cells, and CD16+CD56+ NK cells; (B) Rate of detectability of cell-free virus using single-copy assay, in CSF and plasma and cell-associated (CA) HIV-1 RNA and DNA in CSF cells and PBMC, respectively; (C) comparison of HIV-1 CA-RNA copies, normalized per 106 CD4 T cells, for CSF cells and PBMC; (D) comparison of HIV-1 CA-DNA copies, normalized per 106 CD4 T cells, for CSF cells and PBMC; (E) numbers of CSF cells and PBMC used for extractions; (F) correlation of copy numbers of HIV-1 CA-RNA vs. DNA in CSF cells; (G) correlation of copy numbers of HIV-1 CA-RNA vs. DNA in CSF cells; and (H) correlation of copy numbers of HIV-1 CA-RNA in CSF cells vs. HIV-1 CA-RNA in PBMC.
Fig. 2.
Fig. 2.
Associations of 1H MRS with HIV-1 CA-RNA transcripts and DNA copies. (A) HIV-1 CA-RNA and DNA associations with 1H MRS voxel composite scores. Univariate associations between log-transformed HIV-1 CA-RNA and DNA copy numbers and age-corrected 1H MRS voxel composite z-scores are presented. Asterisk denotes statistical significance at * P < 0.05, **P < 0.01. For the posterior cingulate cortex (PCC)/CSF HIV-1 CA-RNA model, addition of early vs. late initiation of suppressive ART at step 2 led to a small decrease in the strength of the association between the PCC composite score and CSF HIV-1 CA-RNA such that the effect was no longer statistically significant (Std β = −0.53, P = 0.06), and correlations of HIV-1 CA-RNA in CSF cells with: (B) frontal white matter (FWM) composite score; (C) FWM NAA/H2O; (D) PCC composite score; (E) PCC NAA/H2O; (F) Right caudate nucleus composite score; (G) Caudate nuclei NAA/H2O. Nonparametric or parametric correlations were selected depending on whether the distribution was normal.
Fig. 3.
Fig. 3.
Comparison of CA HIV-1 RNA transcripts in CD4+T cells and monocytes in PBMCs. (A) Rate of detectability of cell-associated (CA) HIV-1 RNA and DNA in highly purified CD14+ monocytes from PBMC; (B) number of highly purified CD14+ monocytes and number of CD4+ T cells from CD14-neg PBMC used for extractions for the Double R assay; (C) relative contribution of CD4 T cells and CD14+ monocytes to the total number of CA HIV-1 RNA transcripts from PBMC; (D) comparison of CA HIV-1 RNA copy numbers in CD14+ monocytes from PBMC vs. CD4 T cells in PBMC, in individual patient samples; (E) consistent results for CA HIV-1 RNA from CSF cells, PBMC CD4 T cells, and PBMC CD14+ monocytes, for longitudinal follow-up samples from two participants.
Fig. 4.
Fig. 4.
Memory CD4+ T cell subsets in CSF cells vs. PBMCs. (A) comparisons of % of CXCR3+CD49d+ß7-negative cells in CD4 T cells from CSF vs. PBMC; % of CCR5+ cells in CD4 T cells from CSF vs. PBMC; and % of combined CD38+ and/or HLA-DR+ cells in CD4 T cells from CSF vs. PBMC, and (B) proposed model of directed migration of CXCR3+ CD4+ T cells from blood, including HIV-infected cells, into CSF and CNS parenchyma, due to elevated concentrations of CXCL10 (IP-10), which are in turn due to detection of HIV RNA and viral proteins by myeloid cells in the brain parenchyma.
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