Endogenous retroelement expression in the gut microenvironment of people living with HIV-1

Abstract

Background: Endogenous retroelements (EREs), including human endogenous retroviruses (HERVs) and long interspersed nuclear elements (LINEs), comprise almost half of the human genome. Our previous studies of the interferome in the gut suggest potential mechanisms regarding how IFNb may drive HIV-1 gut pathogenesis. As ERE activity is suggested to partake in type 1 immune responses and is incredibly sensitive to viral infections, we sought to elucidate underlying interactions between ERE expression and gut dynamics in people living with HIV-1 (PLWH).

Methods: ERE expression profiles from bulk RNA sequencing of colon biopsies and PBMC were compared between a cohort of PLWH not on antiretroviral therapy (ART) and uninfected controls.

Findings: 59 EREs were differentially expressed in the colon of PLWH when compared to uninfected controls (padj <0.05 and FC ≤ -1 or ≥ 1) [Wald's Test]. Of these 59, 12 EREs were downregulated in PLWH and 47 were upregulated. Colon expression of the ERE loci LTR19_12p13.31 and L1FLnI_1q23.1s showed significant correlations with certain gut immune cell subset frequencies in the colon. Furthermore L1FLnI_1q23.1s showed a significant upregulation in peripheral blood mononuclear cells (PBMCs) of PLWH when compared to uninfected controls suggesting a common mechanism of differential ERE expression in the colon and PBMC.

Interpretation: ERE activity has been largely understudied in genomic characterizations of human pathologies. We show that the activity of certain EREs in the colon of PLWH is deregulated, supporting our hypotheses that their underlying activity could function as (bio)markers and potential mediators of pathogenesis in HIV-1 reservoirs.

Funding: US NIH grants NCI CA260691 (DFN) and NIAID UM1AI164559 (DFN).

Keywords: Endogenous retroelement (ERE); Human endogenous retrovirus (HERV); Human immunodeficiency virus type I (HIV-I); Interferons; Long interspersed nuclear element (LINE).

Fig. 1

HIV-1 status influences endogenous retroelement expression in the gut microenvironment. Heatmap demonstrating the normalized per sample abundances of EREs following the preprocessing filtering of reads in uninfected controls vs PLWH. ERE reads were filtered to possess at least 2 reads in 10% of the total samples for quality assurance (a). Heatmap demonstrating the normalized per sample abundances differentially expressed EREs between uninfected control and PLWH gut biopsies. (b). Volcano plot demonstrating the average fold change and adjusted p value between uninfected control and PLWH gut biopsies. (c). [Uninfected controls (red) (n = 13) were compared against PLWH (blue) (n = 19); all statistics were performed in DESEQ using the Wald's Test; adjusted p values were calculated using default parameters for a Benjamini-Hochberg correction].

Fig. 2

Correlation between gut endogenous retroelement expression and viral load in people living with HIV-1. Scatterplot demonstrating the viral load from plasma on the X axis and normalized expression of L1FlnI_1q23.1s in colon biopsies on the Y axis (a) [95% CI = 0.14–0.79, displayed p value was calculated using Spearman's correlation coefficient]. Normalized expression of L1FlnI_1q23.1s in colon biopsies from uninfected controls vs PLWH (b) [F = 0.27, num df = 12, denom df = 18, p-value = 0.027; displayed p value was generated using an unpaired t test]. Normalized expression of L1FLnI_1q23.1s in PBMCs from uninfected controls vs PLWH (c) [F = 0.22, num df = 12, denom df = 18, p-value = 0.011; displayed p value was generated using an unpaired t test]. Interferome array of L1FlnI_1q23.1s expression following IFN-a1, -a2, -a5, -a8, -a14, and -b treated gut CD4+ T cells when compared to mock (m) treatment (d) [Bartlett's K-squared = 6.90, df = 6, p-value = 0.33; One-way ANOVA performed for significance and padj values for individual comparisons were determined via Bonferroni corrected t testing for multiple comparisons]. IGV representation of L1FlnI_1q23.1s locus provided by the Telescope annotation alongside the Refseq and Gencode annotations. StringTie reconstructed transcriptomes demonstrate putative transcripts present in sequencing collected from the colon of uninfected controls and PLWH individuals (e). Scatterplot demonstrating the viral load from plasma on the X axis and normalized expression of LTR19_12p13.31 in colon biopsies on the Y axis (f) [95% CI = 0.14–0.82, displayed p value was calculated using Spearman's correlation coefficient]. Normalized expression of LTR19_12p13.31 in colon biopsies from uninfected controls vs PLWH (g) [F = 0.15, num df = 12, denom df = 18, F test p value = 0.0020; displayed p value was generated using an unpaired t test]. Normalized expression of LTR19_12p13.31 in PBMCs from uninfected controls vs PLWH (h) [F = 2.72, num df = 12, denom df = 18, p value = 0.053; displayed p value was generated using an unpaired t test]. Interferome array of LTR19_12p13.31 expression following IFN-a1, -a2, -a5, -a8, -a14, and -b treated gut CD4+ T cells when compared to mock (m) treatment (i) [Bartlett's K-squared = 2.64, df = 6, p value = 0.85; One-way ANOVA performed for significance and padj values for individual comparisons were determined via Bonferroni corrected t testing for multiple comparisons]. IGV representation of LTR19_12p13.31 locus provided by the Telescope annotation alongside the Refseq and Gencode annotations. StringTie reconstructed transcriptomes demonstrate putative transcripts present in sequencing collected from the colon of uninfected controls and PLWH individuals (j). [a1 (n = 3), a2 (n = 3), a5 (n = 3), a8 (n = 3), a14 (n = 3), and b (n = 3) were compared to m (n = 3); uninfected controls (red) (n = 13) were compared against PLWH (blue) (n = 19); shaded regions in scatterplots represent a 95% CI; Tukey box and whisker plots represent all replicates with small dots, median expression with a midline, and mean with an enlarged dot].

Fig. 3

Correlation between LTR19_12p13.31 and L1FLnI_1q23.1s expression and immune signatures of the gut microenvironment in people living with HIV-1. Scatterplot demonstrating the CD4+ T Cells per gram on the X axis and normalized expression of LTR19_12p13.31 in colon biopsies on the Y axis (a) [Uninfected controls (red) (n = 13) 95% CI = −0.69 to 0.69, displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 19) 95% CI = −0.31 to 0.65, displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the CD4+ T Cells per gram on the X axis and normalized expression of L1FLnI_1q23.1s in colon biopsies on the Y axis (b) [Uninfected controls (red) (n = 13) 95% CI = −0.52 to 0.60; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 19) 95% CI = −0.3 to 0.56; displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the CD8+ T Cells per gram on the X axis and normalized expression of LTR19_12p13.31 in colon biopsies on the Y axis (c) [Uninfected controls (red) (n = 13) 95% CI = −0.68 to 0.45; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 19) 95% CI = −0.13 to 0.74; displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the CD8+ T Cells per gram on the X axis and normalized expression of L1FLnI_1q23.1s in colon biopsies on the Y axis (d) [Uninfected controls (red) (n = 13) 95% CI = −0.75 to 0.51; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 19) 95% CI = −0.07 to 0.71; displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the IFNy + CD8+ T Cells per gram on the X axis and normalized expression of LTR19_12p13.31 in colon biopsies on the Y axis (e) [Uninfected controls (red) (n = 9) 95% CI = −0.98 to 0.22; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 18) 95% CI = −0.38 to 0.70; displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the IFNy + CD8+ T Cells per gram on the X axis and normalized expression of L1FLnI_1q23.1s in colon biopsies on the Y axis (f) [Uninfected controls (red) (n = 9) 95% CI = −0.84 to 0.62; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 18) 95% CI = −0.36 to 0.54; displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the CD11c + mDCs per gram on the X axis and normalized expression of LTR19_12p13.31 in colon biopsies on the Y axis (g) [Uninfected controls (red) (n = 13) 95% CI = −0.84 to 0.07; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 19) 95% CI = −0.24 to 0.65; displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the CD11c + mDCs per gram on the X axis and normalized expression of L1FLnI_1q23.1s in colon biopsies on the Y axis (h) [Uninfected controls (red) (n = 13) 5% CI = −0.48 to 0.70; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 19) 844; 95% CI = −0.43 to 0.64; displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the pDCs per gram on the X axis and normalized expression of LTR19_12p13.31 in colon biopsies on the Y axis (i) [Uninfected controls (red) (n = 11) 95% CI = −0.84 to 0.38; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 17) 95% CI = 0.37–0.93; displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the pDCs per gram on the X axis and normalized expression of L1FLnI_1q23.1s in colon biopsies on the Y axis (j) [Uninfected controls (red) (n = 11) 95% CI = −0.61 to 0.77; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 17) 95% CI = −0.35 to 0.75; displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the CD1c-mDCs per gram on the X axis and normalized expression of LTR19_12p13.31 in colon biopsies on the Y axis (k) [Uninfected controls (red) (n = 9) 95% CI = −1.00 to 0.11; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 16) 95% CI = −0.08 to 0.87; displayed p value was calculated using Spearman's correlation coefficient]. Scatterplot demonstrating the CD1c-mDCs per gram on the X axis and normalized expression of L1FLnI_1q23.1s in colon biopsies on the Y axis (l) [Uninfected controls (red) (n = 9) 95% CI = −0.88 to 0.83; displayed p value was calculated using Spearman's correlation coefficient; PLWH (blue) (n = 16) 95% CI = −0.44 to 0.75; displayed p value was calculated using Spearman's correlation coefficient]. [Shaded regions in scatterplots represent a 95% CI].