Abstract
Airway exposure of lupus-prone NZBWF1 mice to crystalline silica (cSiO2), a known trigger of human autoimmune disease, elicits sterile inflammation and alveolar macrophage death in the lung that, in turn, induces early autoimmune onset and accelerates lupus progression to fatal glomerulonephritis. Dietary supplementation with docosahexaenoic acid (DHA), a marine ω-3 polyunsaturated fatty acid (PUFA), markedly ameliorates cSiO2-triggered pulmonary, systemic, and renal manifestations of lupus. Here, we tested the hypothesis that DHA influences both cSiO2-induced death and efferocytotic clearance of resultant cell corpses using three murine macrophage models: (i) primary alveolar macrophages (AM) isolated from NZBWF1 mice; (ii) self-renewing AM-like Max Planck Institute (MPI) cells isolated from fetuses of C57BL/6 mice, and (iii) RAW 264.7 murine macrophages, a virus-transformed cell line derived from BALB/c mice stably transfected with the inflammasome adaptor protein ASC (RAW-ASC). Incubation with cSiO2 at 25 and 50 μg/ml for 6 h was found to dose-dependently induce cell death (p < 0.05) in all three models as determined by both acridine orange/propidium iodide staining and release of lactate dehydrogenase into cell culture supernatant. Pre-incubation with DHA at a physiologically relevant concentration (25 μM) significantly reduced cSiO2-induced death (p < 0.05) in all three models. Cell death induction by cSiO2 alone and its suppression by DHA were primarily associated with caspase-3/7 activation, suggestive of apoptosis, in AM, MPI, and RAW-ASC cells. Fluorescence microscopy revealed that all three macrophage models were similarly capable of efferocytosing RAW-ASC target cell corpses. Furthermore, MPI effector cells could likewise engulf RAW-ASC target cell corpses elicited by treatment with staurosporine (apoptosis), LPS, and nigericin (pyroptosis), or cSiO2. Pre-incubation of RAW-ASC target cells with 25 μM DHA prior to death induced by these agents significantly enhanced their efferocytosis (p < 0.05) by MPI effector cells. In contrast, pre-incubating MPI effector cells with DHA did not affect engulfment of RAW-ASC target cells pre-incubated with vehicle. Taken together, these findings indicate that DHA at a physiologically relevant concentration was capable of attenuating macrophage death and could potentiate efferocytosis, with the net effect of reducing accumulation of cell corpses capable of eliciting autoimmunity.
Highlights
Systemic lupus erythematosus is an autoimmune disease characterized by the loss of immunological tolerance and generation of a diverse pathogenic autoantibody repertoire
The effects of docosahexaenoic acid (DHA) at a physiologically relevant concentration (25 μM) on cSiO2-induced cell death were assessed in three different macrophage models using the experimental design depicted in Figure 1. cSiO2 at 50 μg/ml significantly induced death of primary alveolar macrophages (AMs) compared to vehicle control (Veh)-treated cells as reflected by increased Propidium Iodide (PI) staining (Figures 2A,B) and Lactate Dehydrogenase (LDH) release (Figure 2C)
Max Planck Institute (MPI) cells were more sensitive to cSiO2’s cytotoxic effects with concentrations of 25 and 50 μg/ml inducing significantly increases in PI positive cell corpses (Figure 2B and Supplementary Figure 1A) and similar trends observed for LDH responses (Figure 2C)
Summary
Systemic lupus erythematosus (lupus) is an autoimmune disease characterized by the loss of immunological tolerance and generation of a diverse pathogenic autoantibody repertoire. This disease often manifests as alternating episodes of remission and flaring, eventually causing irreversible damage to multiple organs including the kidney, skin, heart, and brain. The pulmonary response is associated sequentially with robust proinflammatory cytokine, chemokine, and interferon-related gene expression, ectopic lymphoid tissue development, and diverse autoantibody production, driving systemic autoimmune disease progression and culminating in end-stage glomerulonephritis [20, 22, 23]
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