Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension

Abstract

Background: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies.

Methods: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat.

Results: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1β, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6.

Conclusions: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

Keywords: SAM domain and HD domain-containing protein 1 (SAMHD1); deoxyuridine triphosphate nucleotidohydrolase (dUTPase); human endogenous retrovirus K (HERV-K); pulmonary arterial hypertension (PAH); tertiary lymphoid tissue.

Figure 1. SAMHD1 a target antigen in immune complexes in PAH lungs

(A) C1q immunoprecipitation followed by liquid chromatography tandem mass spectrometry identified target antigens of immune complexes in PAH (n=3) and control (n=3) lungs. Targets are ranked by q value according to the SAM statistic and all were within the false discovery rate (FDR) of 5%. (B) SAMHD1 immune complexes measured by ELISA in PAH (n=16) and control (n=12) lungs. ****P<0.0001 by Welch test. (C) Representative sections from a PAH lung show tertiary lymphoid tissue (tLT), characterized by positive immunoreactivity to markers of T cells (CD3), B cells (CD19), plasma cells (rough endoplasmic reticulum-associated protein p63) and follicular dendritic cells (FDC; 120 kDa FDC protein). (D) Number of PAs with associated tertiary lymphoid tissue relative to total PAs was calculated as a percentage from lung tissue sections in each control (n=15) and each PAH patient (n=13) and controls. ****P<0.0001 by Mann-Whitney test. (E) SAMHD1 immunoreactive foci in tertiary lymphoid tissue detected by applying recombinant GST-tagged SAMHD1 protein to lung tissue sections, followed by an anti-GST-FITC conjugated antibody Negative controls were treated with PBS, followed by anti-GST-FITC conjugated antibody. Nuclei were stained by DAPI (blue). Ranges represent mean ± SEM (B) and median with interquartile range (D). Closed symbols (PAH), open symbols (controls), closed triangles hereditary PAH (HPAH).

Figure 2. Elevated SAMHD1 in PAH lung cells and in circulating classical dendritic cells (cDC)

(A) Representative western immunoblot with densitometric quantitation of SAMHD1 in lung lysates assessed in PAH (n=5) vs. control (n=5) lungs. **P<0.01 by Student’s t-test. (B) Representative immunohistochemistry of SAMHD1 in pulmonary artery (PA) from a donor (control) lung, and from a PA of similar size and at similar airway level from a lung of a PAH patient. Below, percent nuclei that stained for SAMHD1 in all arteries in a lung section in PAH (n=6) vs. control (n=6) lungs, calculated using the ImmunoRatio program. The dashed line indicates the vessel boundary including the adventitia, within which % SAMHD1 positive cells was calculated. **P<0.01 by Student’s t-test. (C) Confocal microscopic images of sections immunolabeled with SAMHD1 (green), and four lineage markers (red): left to right, vWF (endothelial cells), CD11c (dendritic cells), CD68 (macrophages) and CD3 (T cells). Nuclei were stained with DAPI (blue). Dashed line indicates vessel boundary. (D) SAMHD1 assessed by CyTOF in circulating classical dendritic cells (cDCs) from PAH patients (n=10) or controls (n=8). Data are shown as the calculated difference of inverse hyperbolic sine medians between control and PAH samples (Arcsinh Ratio). **P<0.01 by Mann-Whitney test. Ranges represent mean ± SEM (A, B) and median with interquartile range (D). Closed symbols (PAH), open symbols (controls), closed triangles hereditary PAH (HPAH).

Figure 3. Elevated HERV-K and HERV-K dUTPase detected in lungs and circulating monocytes from PAH vs. controls

(A) HERV species in lung tissue from PAH patients (n=4) and controls (n=4) by metagenomic sequencing described in Methods. MSRV, multiple sclerosis-associated retrovirus. (B) HERV-K(II) envelope and dUTPase mRNA by qPCR in lung extracts from PAH patients (n=10) and controls (n=10). **P<0.01 by Mann-Whitney test. (C) Confocal microscopy images of representative lung sections from a PAH patient and a control, show cells immunolabeled for HERV-K envelope protein or HERV-K dUTPase (green), macrophages (CD68⁺, red) and nuclei (DAPI, blue). Dashed line indicates vessel boundary. Elastin auto-fluorescence appears pink. (D) HERV-K dUTPase mRNA in circulating monocytes from PAH patients (n=5) vs. controls (n=5). **P<0.01 by Student’s t-test. Ranges represent mean ± SEM (D) and median with interquartile range (B). Closed symbols are PAH patients, open symbols are controls, closed triangles represent hereditary PAH (HPAH) patients.

Figure 4. HERV-K dUTPase increases SAMHD1, cytokines in enriched monocytes and pulmonary arterial endothelial cells (PAEC), and activates B cells

(A, B) Enriched monocytes from PBMC (A) or PAEC (B) from healthy donors were treated with recombinant HERV-K dUTPase (0.1, 1 or 10 μg/mL) for 24 hr, and SAMHD1 was assessed by western immunoblot. (C, D) TNFα, IL1β and IL6 measured by ELISA in medium of enriched monocytes (C) or PAEC (D), treated with 0.1 or 1 μg/mL HERV-K dUTPase. (E) CD69⁺ B cells, and (F) signaling molecules assessed by CyTOF following HERV-K dUTPase (10 μg/mL for 24 hr). Signal induction was calculated as the difference of inverse hyperbolic sine medians between untreated (Control, Con) and HERV-K dUTPase-treated samples (arcshinh ratio). Ranges represent mean ± SEM of n=4 different experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Student’s t-test (B, E), or by one-way ANOVA and post hoc Dunnett’s test (A, C and D). In (F), Bonferroni-adjusted P-value (P=7.14X10⁻³) is applied to response with arcsinh ratio > |0.2|).

Figure 5. HERV-K dUTPase increases apoptosis in pulmonary arterial endothelial cells (PAEC) in an IL6 independent manner, is induced in monocytes by LPS and is elevated in iPSC from PAH patients vs. controls

(A) PAEC were treated with 10 μg/mL HERV-K dUTPase and apoptosis was assessed by Caspase-Glo 3/7 assay following overnight serum withdrawal (n=4). (B) PAEC were pre-treated with neutralizing IL6 antibody (IL6 Ab) or isotype control (Con) before HERV-K dUTPase treatment (n=4). (C) HERV-K dUTPase mRNA by qPCR in monocytes stimulated with LPS 1μg/ml (n=3). (D) HERV-K dUTPase mRNA by qPCR in iPSC from PAH patients (n=7) and controls (n=7). Ranges represent mean ± SEM. *P<0.05, ***P<0.001, ****P<0.0001 by Student’s t-test (A, C, D). In (B), **P<0.01, HERV-K dUTPase vs. Vehicle treatment, by one-way ANOVA and post hoc Tukey test. Closed symbols (PAH), open symbols (controls), closed triangles hereditary PAH (HPAH).

Figure 6. HERV-K dUTPase causes pulmonary hypertension in a rat

Adult male Sprague Dawley rats (7 wks, 180–200 g) were treated with HERV-K dUTPase (0.2 mg/kg), or saline (vehicle) once a week, for three weeks (LHS) or were pre-treated with the VEGF receptor 2 blocker SU5416 (20 mg/kg) (RHS). Pulmonary and cardiac functions were evaluated on day 21. (A) Pulmonary artery acceleration time (PAAT). (B) Right ventricular systolic blood pressure (RVSP). (C) Right ventricular hypertrophy (RVH). (D) Confocal microscopic images of lung sections of treated and control rats, immunolabeled for αSMA (green, smooth muscle cell marker), and vWF (red, endothelial cells) on the left, with the % of muscularized distal vessels on the right. (E, F) IL6 levels in lungs, by ELISA (E) and IL6 staining by IHC (F). Saline (n=8) or HERV-K dUTPase (n=8), SU5416 + Saline (n=13) or SU5416 + HERV-K dUTPase (n=10). Ranges represent mean ± SEM (A, C, D, E and B, left) and median with interquartile range (B, right). *P<0.05 **P<0.01 and ****P<0.0001 by Student’s t-test in (A, C, D and E) or by Mann Whitney test (B).

Figure 7. Proposed model for the role of HERV-K and SAMHD1 in PAH

The endogenous retrovirus HERV-K is expanded, possibly as a result of an environmental or genotoxic stress. The product, HERV-K dUTPase, and the subsequent activation of vascular, inflammatory and immune cells lead to adverse vascular remodeling and PAH.