Heterozygous OAS1 gain-of-function variants cause an autoinflammatory immunodeficiency

Abstract

Analysis of autoinflammatory and immunodeficiency disorders elucidates human immunity and fosters the development of targeted therapies. Oligoadenylate synthetase 1 is a type I interferon-induced, intracellular double-stranded RNA (dsRNA) sensor that generates 2'-5'-oligoadenylate to activate ribonuclease L (RNase L) as a means of antiviral defense. We identified four de novo heterozygous OAS1 gain-of-function variants in six patients with a polymorphic autoinflammatory immunodeficiency characterized by recurrent fever, dermatitis, inflammatory bowel disease, pulmonary alveolar proteinosis, and hypogammaglobulinemia. To establish causality, we applied genetic, molecular dynamics simulation, biochemical, and cellular functional analyses in heterologous, autologous, and inducible pluripotent stem cell-derived macrophages and/or monocytes and B cells. We found that upon interferon-induced expression, OAS1 variant proteins displayed dsRNA-independent activity, which resulted in RNase L-mediated RNA cleavage, transcriptomic alteration, translational arrest, and dysfunction and apoptosis of monocytes, macrophages, and B cells. RNase L inhibition with curcumin modulated and allogeneic hematopoietic cell transplantation cured the disorder. Together, these data suggest that human OAS1 is a regulator of interferon-induced hyperinflammatory monocyte, macrophage, and B cell pathophysiology.

Fig. 1.. A polymorphic autoinflammatory immunodeficiency with de novo heterozygous OAS1 variants.

(A) Ancestry of patient 1–6 (abbreviated P1–6 throughout the figures) and electropherograms [fibroblasts (patient 1–4), PBMCs (patient 5), and buccal swabs (patient 6)] of de novo germline heterozygous OAS1 variants. (B) Clinical photographs of facial rash (patient 6) and skin erosions (patient 4). (C) Lung histology (patient 1) of PAP with granular material, cholesterol cleft-like cracks (upper image, bar = 250 μm), and foamy alveolar macrophages (lower image, bar = 50 μm). (D) Skin histology (patient 3) with intra- and subepidermal blister formation, epidermal lymphocytes, and dermal chronic inflammation (upper image, bar = 500 μm); duodenal histology (patient 3) with marked villous atrophy, crypt hyperplasia and epithelial regeneration (lower image, bar = 100 μm). (E) Chest radiography (patient 1) with PAP 14 days before (upper image) and resolution 1 year after HCT (lower image). (F) OAS superfamily phylogenetic analysis indicating conservation of A76, C109, V121, and L198 for the full OAS1 and the human OAS1 subclade. (G) Consensus and sequence logos within the human OAS1 subclade surrounding A76, C109, V121, and L198. (H) Pseudo-color plots, overlay histograms, and summary graphs of PBMCs, monocytes (CD14⁺), B-cells (CD19⁺), and T-cells (CD3⁺) either unstimulated (Med) or after IFNα-, CpG- and CD3/CD28-stimulation showing time-dependent changes in median intracellular OAS1 expression. (I) Overlay histograms and summary graph of intracellular OAS1 expression in naïve (IgD⁺CD27^-, Naive), un-switched memory (IgD⁺CD27⁺, UM), class-switched memory (IgD^-CD27⁺, SM) B-cells, and plasmablasts (CD27⁺CD38⁺, PB) in unstimulated (Med) and CpG-stimulated PBMCs. For H and I, summary graphs of 6 different OAS1-WT donors, patient 1 (A76V) and patient 6 (C109Y) from 4 independent experiments (error bars, mean ± s.d.) are shown.

Fig. 2.. Functional and transcriptomic alteration in OAS1-A76V and OAS1-C109Y monocyte, B- cells, and T-cells.

(A) Pseudo-color plots showing B-cell proliferation of unstimulated (Med) and CpG-stimulated PBMCs from OAS1-WT, A76V, and C109Y after 5 days by CFSE-dilution and up-regulation of CD27. Graphs show summary of B-cell proliferation percentages for 6 different OAS1-WT, A76V and C109Y from 5 independent experiments. (B) Pseudo-color plots showing B-cell differentiation of naïve B-cells (IgD⁺CD27^-, Naive) into un-switched memory (IgD⁺CD27⁺, UM), class-switched memory (IgD^-CD27⁺, SM) B-cells, and plasmablasts (CD27⁺CD38⁺, PB)òf unstimulated (Med) and CpG-stimulated PBMCs from OAS1-WT, A76V, and C109Y after 5 days. Graphs show summary of B-cell differentiation for 6 different OAS1-WT, A76V and C109Y from 5 independent experiments. (C) Histogram plots of cleaved PARP-1 (cl.PARP) in unstimulated and medium- (Med), CpG-, and CD3/CD28–48h-stimulated PBMCs, monocytes (CD14⁺), B-cells (CD19⁺), and T-cells (CD3⁺), and summary of percentages of cleaved PARP for 4 different OAS1-WT, A76V, and C109Y from 4 independent experiments. (D) Pseudo-color plots of CD25 expression and CFSE-dilution on CD4⁺ T-cells in a mixed lymphocyte reaction after allogeneic B-cell stimulation of 3 different allogeneic OAS1-WT donors and A76V without (Med) and after stimulation with anti-CD3 and anti-CD3/CD28 for 5 days. Summary graph of CD4⁺ T-cell proliferation percentages and median CD25 expression for 4 different OAS1-WT (gray) and A76V (red) from 3 independent experiments. (E) Venn diagram intersection of DEGs (adjusted p-value <0.05 and log₂FC>0.5) in monocytes, B-cells, and T-cells from 3 OAS1-WT and A76V. (F) GO-term enrichment analysis of DEGs specific for monocytes, B-cells, and T-cells (non-redundant terms related to immune function with Fisheŕs exact P-value <0.05 containing >10 genes; a complete GO-term list is presented in Tab. S5). (A-D) Significance levels are calculated with two-sided Welchś t-test and indicated in the summary graphs (NS = non-significant).

Fig. 3.. Interferon-α induced RNase L-dependent dysfunction of monocyte and iPSC-derived macrophages.

(A) Overlay histograms of phosphorylated STAT5 (pSTAT5) in monocytes of unstimulated (Med), GM-CSF-, IL-3-, and pervanadate- (PV) stimulated PBMCs and summary of median pSTAT5 from 5 different OAS1-WT (gray), A76V (red), and C109Y (blue) from 3 independent experiments. (B) Fluorescence microscopy of OAS1-WT and L198V alveolar macrophages (green nuclear staining) and phagocytosed PE-latex beads (red). (C) Bright-field microscopy of OAS1-WT, L198V, KI-L198V, and KI-C109Y iPSC-MΦ either unstimulated or IFNα-stimulated (−/+IFNα) for 48 hours, and summary graph of proportions of floating single cells (FSC) of total live iPSC-MΦ unstimulated (black) or IFNα-stimulated (red) from 3 independent experiments. (D) Pseudo-color plots of OAS1-WT and L198V iPSC-MΦ unstimulated or IFNα-stimulated for 7 days. (E) Proportions of floating single cells (FSC) of total live OAS1-WT, L198V, KI-L198V iPSC-MΦ, and KI-C109Y iPSC-MΦ unstimulated (black), IFNα- (red), and curcumin (orange) stimulated/inhibited for 48 hours from 3 independent experiments. (F) Histogram overlays of PE-latex beads phagocytosed by OAS1-WT and L198V iPSC-MΦ unstimulated (black) or IFNα- (red) stimulated for 48 hours and summary graph of 2 OAS1-WT and 2 L198V clones analyzed in 3 independent experiments. (G) Relative expression levels of surface scavenger receptors of OAS1-WT (white) and L198V (red) iPSC-MΦ unstimulated and IFNα-stimulated for 48 hours from 3 independent experiments. (H) MA-plot visualization of DEGs (magenta) with a FDR <0.05 between unstimulated and 48 hours IFNα-stimulated OAS1-WT and L198V iPSC-MΦ. (I) KEGG-based GSEA of down-regulated DEG in IFNα-stimulated L198V iPSC-MΦ as compared to OAS1-WT. Significance levels are calculated with two-sided paired t-test and indicated in the summary graphs (ns = non-significant, * p<0.05, ** p<0.005, error bars, mean ± s.d.).

Fig. 4.. OAS1-GOF 2–5A synthetase activity- and RNase L-dependent cellular RNA degradation, protein translational arrest, and apoptosis.

(A) RNA chip analysis from OAS1-WT, A76V, and C109Y monocytes (CD14⁺), B-cells (CD19⁺), and T-cells (CD3⁺) of total RNA degradation and (B) summary graph of RIN from 5 independent experiments (NS = non-significant). (C) In vitro activity for OAS1-WT and OAS1-MUT (A76V, C109Y, V121G, L198V) proteins in the absence of poly(rI:rC) dsRNA after 60 minutes (error bars, mean ± s.e.m). (D) Chromogenic analysis of reaction time courses for purified OAS1-WT (white bars), A76V (black bars), and L198V (purple bars) protein in the absence of poly(rI:rC) dsRNA for a range of protein concentrations (error bars, mean ± s.e.m). (E) 2–5A biosensor analysis of unstimulated and poly(rI:rC) dsRNA-stimulated HEK293T cells transiently transfected with OAS1-WT and OAS1-MUT (error bars, mean ± s.d.). (F) RNA chip analysis of total RNA isolated from 1205Lu melanoma RNase L wild-type (WT) and RNase L knockout (KO) cell lines untransfected (None) and transiently transfected with OAS1-WT, OAS1-MUT, or poly(rI:rC) (pIC). (G) Expression levels of OAS1-WT or OAS1-MUT in transfected 1205Lu melanoma wild-type (WT) and RNase L knockout (KO) cell lines determined by eGFP co-expression 48h after transfection (error bars, mean ± s.d.). (H and I) Translational activity (GFP-positive/GFP-negative) and frequency of dead (Annexin-V⁺) cells 48h after transfection (error bars, mean ± s.d., * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Fig. 5.. dsRNA-independent OAS1-GOF 2–5A synthetase activity favored by allosteric loss of structural constraints.

MD simulation analysis of OAS-WT and OAS-MUT: (A) overall protein root-mean-square deviation (RMSD) over the 100 ns MD production run, (B) residue backbone atom root-mean-square fluctuation (RMSF), and (C) difference RMSF (OAS-WT subtracted from each OAS-MUT). Individual proteins are as noted in the plot legends (left to right): A76V (blue), C109Y (orange), V121G (purple) and L198V (green); OAS-WT (black) is shown in panels A and B for comparison to each OAS-MUT. Shaded regions on plots in panel B are the error in each value (SD). In panel C, dotted lines denote two standard deviations (+/− 2σ) from the average difference RMSF for each protein (i.e., the most significant changes in residue dynamics). (D) Residues with most significant changes in dynamics mapped onto OAS1 structure (shown in the dsRNA-bound conformation; PDB code 4RWN) as Cα spheres; color coding denotes increased dynamics (> 2σ; red) and decreased dynamics (< −2σ; blue) compared to OAS-WT. (E) Locations of the OAS-MUT substituted residues shown in the same views as panel D. (F) Model for common action of OAS1-GOF variants. OAS1-WT (left) strictly regulated by dsRNA binding-induced conformational changes, whereas OAS1-MUT amino acid substitutions (right) relieve this strict control by allosteric induction of changes around the enzyme catalytic center (red dotted lines) and mirroring changes that occur upon dsRNA binding (blue dotted lines). These changes promote low-level GOF activity in the absence of dsRNA.