Type I IFN drives unconventional IL-1β secretion in lupus monocytes

Abstract

Opsonization of red blood cells that retain mitochondria (Mito⁺ RBCs), a feature of systemic lupus erythematosus (SLE), triggers type I interferon (IFN) production in macrophages. We report that monocytes (Mos) co-produce IFN and mature interleukin-1β (mIL-1β) upon Mito⁺ RBC opsonization. IFN expression depended on cyclic GMP-AMP synthase (cGAS) and RIG-I-like receptors' (RLRs) sensing of Mito⁺ RBC-derived mitochondrial DNA (mtDNA) and mtRNA, respectively. Interleukin-1β (IL-1β) production was initiated by the RLR antiviral signaling adaptor (MAVS) pathway recognition of Mito⁺ RBC-derived mtRNA. This led to the cytosolic release of Mo mtDNA, which activated the inflammasome. Importantly, mIL-1β secretion was independent of gasdermin D (GSDMD) and pyroptosis but relied on IFN-inducible myxovirus-resistant protein 1 (MxA), which facilitated the incorporation of mIL-1β into a trans-Golgi network (TGN)-mediated secretory pathway. RBC internalization identified a subset of blood Mo expressing IFN-stimulated genes (ISGs) that released mIL-1β and expanded in SLE patients with active disease.

Keywords: MxA; NLRP3; inflammasome; monocytes; red blood cells; systemic lupus erythematosus; type I interferon.

Figure 1.. Erythrophagocytic Mo co-expressing IL-1β and ISGs are expanded in active SLE.

A, Confocal image (left) and quantification (right) of classical Mo stained with IL-1β and MxA antibodies (SLE n=20; JDM n=8). Scale bar: 20 μm. B, Two-tailed Pearson’s correlation between SLEDAI and the percentage of IL-1β⁺ ISGs⁺ Mo (n=20). Dashed lines represent 95% confidence intervals. Each dot represents a study participant sample that was processed, stained and analyzed independently. C, Confocal images of secreted mIL-1β from classical Mo sorted from SLE patient. Slides were coated with anti-mIL-1β capture antibody and mitochondria were stained with MitoTracker Deep Red (MTDR). Scale bar: 20 μm. D, Percentages of mIL-1β⁺ MTDR⁺ Mo from SLE patients (n=6). Each dot represents the count from different microscopy fields from a study participant sample that was processed, stained, and analyzed independently. E, Percentage of IL-1β⁻ MxA⁺ Mo and IL-1β⁺ MxA⁺ Mo in SLE patients without (blue, n=8) or with (red, n=6) circulating Mito⁺ RBCs. F, Confocal images of classical Mo isolated from SLE patients showing evidence of erythrophagocytosis. GPA: Glycophorin A. Scale bar: 20 μm. G, Flow cytometry anaylsis, of intracellular GPA, in SLE (n=18) and HD (n=4) blood Mo, gated as CD3⁻ CD19⁻ HLADR⁺ CD11c⁻ CD123⁻ cells.

Figure 2.. Internalization of opsonized Mito⁺ RBCs triggers Type I IFNs and IL-1β production in human Mo.

A, Heat map of differentially expressed genes in blood Mo that phagocytized either Mito⁻ or Mito⁺ RBCs. Data are normalized to Mito⁻ RBCs sample. B, Concentrations of IP-10 and IL-1β in the supernatants of blood Mo cultured with media, Mito⁻ or Mito⁺ RBCs. (n=6). C, Immunoblot analysis of whole cell lysate (WCL) and supernatants (Sup) from blood Mo that were treated as described. One representative of two independent experiments. D, Concentrations of IP-10 and IL-1β in the supernatants of bone marrow (BM) Mo cultured with media, Mito⁻ or Mito⁺ opsonized RBCs. (n=3). E, Heat map of differentially expressed genes in BLaER1 Mo that phagocytized Mito⁻ or Mito⁺ RBCs. Data are normalized to Mito⁻ RBCs sample. F, Concentrations of IP-10 and IL-1β in the supernatants of BLaER1 Mo cultured with media, Mito⁻ or Mito⁺ RBCs. (n=4). G, Immunoblot analysis of WCL and Sup from BLaER1 Mo that were treated as described. One representative of three independent experiments.

Figure 3.. Mito⁺ RBCs-derived NAs trigger cGAS-dependent IFN and NLRP3-dependent IL-1β production in human Mo.

A, Relative mtDNA abundance and Western blot analysis of Mito⁺ RBCs generated with vehicle, ethidium bromide (EtBr; ρ⁰) or chloramphenicol (CAM). (n=3). The experimental scheme is also shown. B, Concentrations of IP-10 in the supernatants of BLaER1 Mo activated with Mito⁺ RBCs generated in the presence of vehicle, EtBr (ρ⁰) or CAM. (n=4). C, Concentrations of IL-1β in the supernatants of Mito⁺ RBCs-activated wild type, Cgas^−/− or Sting1^−/− BLaER1 Mo. (n=3). D, Concentrations of IP-10 in the supernatants of Mito⁺ RBCs-activated wild type or Cgas^−/− BLaER1 Mo. (n=3). E, Concentrations of IP-10 in the supernatants of Mito⁺ RBCs-activated wild type or Sting1^−/− BLaER1 Mo. (n=3). F, Concentrations of IL-1β in the supernatants of Mito⁺ RBCs-activated wild type or Aim2^−/− BLaER1 Mo. (n=3). Concentrations of IL-1β in the supernatants of Mito⁺ RBCs-activated Nlrp^−/− (G), Casp1^−/− (H) or Pycard^−/− (I) BLaER1 Mo. (n=3).

Figure 4.. Mito⁺ RBC-derived mtDNA binds cGAS while Mo-derived mtDNA binds NLRP3.

A, Confocal images of BLaER1 Mo that were treated with media or with 5-ethynyl-2-deoxyuridine (EdU)-labelled Mito⁺ RBCs (Mito⁺ RBCs^Edu) and then stained for the phagolysosomal marker LAMP1. Scale bars, 20 μm. Quantification of cytosolic mtDNA (LAMP1⁻ EdU⁺ speckles per cell) is also shown. (n=20 cells). B, Dot blot-Western blot analysis (left), experimental scheme (middle) and quantification (right) of Mito⁺ RBCs (lanes 1 - 3) or BLaER1 Mo (lanes 2 – 4) labeled with 5-bromo-2-deoxyuridine (BrdU) and then immunoprecipitated (IP) with cGAS or NLRP3 antibodies. As a loading control, cGAS or NLRP3 immunoblot or double-stranded DNA (dsDNA) dot blot (DB) was performed. A.U.: Arbitrary Units. C, Western blot analysis of cytosolic fraction obtained from media, Mito⁺ RBCs- or dsDNA/Lyovec-activated BLaER1 Mo. WCL: whole cell lysate. One representative of two independent experiments. D, Quantification of lactate dehydrogenase (LDH) release (left) or propidium iodide (PI) internalization (right) in BLaER1 Mo activated with Media, Mito⁺ RBCs or dsDNA/Lyovec at different time points. (n=3).

Figure 5.. Mito⁺ RBC-derived mtRNA triggers the release of Mo-derived mtDNA fragments.

A, Experiment scheme and IL-1β concentrations in the supernatants of BLaER1 Mo, generated in the presence of vehicle, EtBr (ρ⁰) or CAM and activated with Mito⁺ RBCs. (n=4). B, Expression of mtDNA-encoded genes in NLRP3 immunocomplexes isolated from Mito⁺ RBC-activated BLaER1 Mo. (n=3). C, Confocal images of EdU-labeled BLaER1 Mo (BLaER1 Mo^Edu) that were treated with medium or Mito⁺ RBCs and then co-stained for the mitochondrial marker TOMM20. Scale bars, 20 μm. Quantification of cytosolic mtDNA (TOMM20⁻ EdU⁺ speckles per cell) is also shown. (n=20 cells). D, Experiment scheme and cytokine concentrations in the supernatants of BLaER1 Mo cultured with Mito⁺ RBCs generated in the presence of Actinomycin D (Mito⁺ RBCs^{Act D}). (n=4). E, Concentrations of IL-1β in the supernatants of Mito⁺ RBCs-activated wild type, Mavs^−/− or Rigi^−/− Ifih1^−/− BLaER1 Mo. (n=3).

Figure 6.. Mito⁺ RBCs induced mIL-1β secretion requires MxA but not pyroptosis or gasdermin-D pores.

A, Western blot analysis of gasdermin-D ~30 KDa N-terminal fragment (GSDMD-N30) in total cell lysate isolated from Mito⁺ RBCs, Poly I:C/Lyovec and Nigericin-activated BLaER1 Mo. B, Quantification of lactate dehydrogenase (LDH) release or propidium iodide (PI) internalization in BLaER1 Mo activated with Media, Mito⁺ RBCs, Poly I:C/Lyovec or Nigericin at different time points. (n=3). C, Western blot analysis of Gsdmd^−/− BLaER1 cells. (left). Concentrations of IL-1β in the supernatants from Mito⁺ RBCs, Poly I:C/Lyovec and Nigericin-activated wild type or Gsdmd^−/− BLaER1 Mo (right). (n=3). D, Concentrations of IL-1β in the supernatants from Mito⁺ RBCs, Poly I:C/Lyovec and Nigericin-activated Blood Mo in the presence of disulfiram. (n=3). E, Confocal images of SLE classical Mo stained for IL-1β and MxA. Scale bar: 20 μm. F, Concentrations of IL-1β in the supernatants from Mito⁺ RBCs, Poly I:C/Lyovec and dsDNA/Lyovec-activated wild type or Mxa^−/− BLaER1 Mo. (n=3). G, Western blot analysis of GSDMD in total cell lysate isolated from Mito⁺ RBCs or Nigericin-activated iPSC Mo. One representative of two independent experiments. H, Flow cytometry analysis of CD14 expression in wild type or Mxa^−/− iPSC at day 9 or 17 post-differentiation into Mo. I, Concentrations of IL-1β in the supernatants from Mito⁺ RBCs or Nigericin-activated wild type or Mxa^−/− iPSC Mo. (n=3).

Figure 7.. MxA oligomerization promotes the translocation of mIL-1β within the TGN.

A, Experimental scheme (left) and Western blot analysis (right) of the microsomal pellet (100k P) and postmicrosomal supernatant (100k S) isolated from Poly I:C/Lyovec-activated BLaER1 Mo. B, Proteinase K (PK) protection assay of the membrane fraction isolated from Poly I:C/Lyovec-activated wild type or Mxa^−/− BLaER1 Mo. TX100: Triton X-100. C, Experimental scheme (top) and Western blot analysis (bottom) of the in vitro proteoliposomes (PtLp)-based transport assay. One representative of two independent experiments. D, Immunoblot analysis of mIL-1β present within liposomes (pellet), or supernatants (sup) after ultracentrifugation of liposomes that were treated with buffer or recombinant human MxA for 0 (control), 30 and 120 min. Densitometry quantification of Western blot band density for mIL-1β release from liposomes is also shown. (n=3). E, Western blot analysis of different fractions collected after sucrose fractionation of microsomal membranes isolated from Poly I:C/Lyovec-activated BLaER1 Mo. One representative of three independent experiments. ER: endoplasmic reticulum; ERGIC: ER-Golgi intermediate compartment. Confocal images of Poly I:C/Lyovec-activated BLaER1 Mo stained for TNG46 and MxA (F) or TGN46 and IL-1β (G). Scale bar: 20 μm.