OLFML3 Promotes IRG1 Mitochondrial Localization and Modulates Mitochondrial Function in Macrophages

Abstract

Olfactomedin-like protein 3 (OLFML3), belonging to olfactomedin (OLF) protein family, has poorly defined functions. Recent studies have reported the functions of OLFML3 in anti-viral immunity and tumorigenesis. In this study, we investigated the roles of OLFML3 in macrophages. In LPS- or Pseudomonas aeruginosa-induced acute lung injury (ALI) mouse model, OLFML3 depletion exacerbated inflammatory response, leading to reduced survival. OLFML3 achieved the in vivo activity by regulating macrophage phagocytosis and migration. Mass spectrometry analysis revealed immunoresponsive gene 1 (IRG1) as an OLFML3-interacting protein. IRG1 is a mitochondrial decarboxylase that catalyzes the conversion of cis-aconitate to itaconate, a myeloid-borne mitochondrial metabolite with immunomodulatory activities. Further investigation showed that OLFML3 could prevent LPS-induced mitochondrial dysfunction in macrophages by maintaining the homeostasis of mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (mtROS) and itaconate-related metabolites. In-depth protein-protein interaction studies showed that OLFML3 could promote IRG1 mitochondrial localization via a mitochondrial transport protein, apoptosis inducing factor mitochondria associated 1 (AIFM1). In summary, our study showed that OLFML3 could facilitate IRG1 mitochondrial localization and prevent LPS-induced mitochondrial dysfunction in macrophages.

Keywords: IRG1; OLFML3; acute Lung Injury; macrophage; mitochondria.

Figure 1

Olfml3 knockout elevates pulmonary inflammation in LPS- or PAO1-stimulated mice. (A) Survival curve of wild-type and Olfml3 knockout mice challenged with 10 mg/kg LPS (n = 11 per group). LPS is intraperitoneally administrated to induce sepsis. (B) Evaluation of LPS- or PAO1-induced pulmonary edema, as determined by lung wet-to-dry ratio. (C) H&E staining of lung sections for analysis of inflammatory cell infiltration upon LPS and PAO1 stimulation. Scale bar, 100 μm. Black arrows indicate infiltrating cells. (D) Analysis of the total cell counts in BALF. (E) Determination of the concentrations of total proteins in BALF using BCA methods. (F) H&E staining of lung sections for analysis of alveolar macrophage depletion in Olfml3^+/+ and Olfml3^-/- mice. Scale bar, 100 μm. (G, H) Analysis of the total cell counts (G) and total proteins (H) in BALF from alveolar macrophage-depleted Olfml3^-/- mice. For B to H, LPS (10 mg/kg) or PAO1 (2×10⁶ colony-forming units, CFU) is intranasally instilled to mice (n = 5 or 6 per group). The data are presented as mean ± SEM of three independent experiments.

Figure 2

Olfml3 knockout reduces the phagocytic and migration functions of macrophages. (A, B) Analysis of the phagocytic function of RAW264.7 (left) and BMDMs (right) using latex beads carrying FITC-labeled IgG using flow cytometry (A) and immunofluorescence (B) experiments. The cells are incubated with the beads for 2 h. (C, D) Evaluation of the effects of OLFML3 on the migration of RAW264.7 (C) and BMDMs (D) in the absence or presence of LPS using transwell assay. At 24 h after 1 μg/mL LPS stimulation, the transmembraned cells are counted from three microscopic fields with 40× magnification. One representative microscopic field of each group is shown on the left (Magnification 20×; Scale bar, 150 μm). The bar graphs show the averaged values of three biological replicates. (E, F) Determination of MCP-1expression at intracellular mRNA (E) and soluble protein (F) levels in RAW264.7 (left) and BMDMs (right), using RT-qPCR and ELISA respectively. The Olfml3^-/- cells are isolated single clones containing gene knockout at both alleles of Olfml3. For E, cells are collected at 4 h post 100 ng/mL LPS stimulation. For F, culture supernatant is collected at 24 h after 100 ng/mL LPS stimulation. The data are the mean ± SEM of three biological replicates. ns, not significant.

Figure 3

Dissection of the pattern of interactions between OLFML3 and IRG1. (A) Identification of IRG1 as an OLFML3-interacting protein in RAW264.7 by mass spectrometry analysis of OLFML3-Myc/Flag co-immunoprecipitated proteins. OLFML3-interacting proteins are enriched using Flag magnetic beads. The top 5 candidate proteins are shown. (B) Schematic presentation of OLFML3 and IRG1 domain organization. (C) Validation of the interactions between OLFML3 and IRG1 in HEK293T cells. OLFML3-Myc/Flag is co-expressed with IRG1-HA, immunoprecipitated (IP) by HA beads and immunoblotted (IB) with Flag antibody. (D) Investigation of IRG1-interacting OLFML3 domains in HEK293T cells. Myc/Flag-tagged OLFML3 constructs are co-expressed with IRG1-HA, immunoprecipitated by HA beads and immunoblotted with Flag antibody. (E) Investigation of OLF domain-interacting IRG1 domains in HEK293T cells. HA tagged IRG1 constructs are co-expressed with OLF-Myc/Flag, immunoprecipitated by HA beads and immunoblotted with Flag antibody. (F) Validation of the interactions between OLFML3-Myc/Flag and endogenous IRG1 in a RAW264.7 cell line stably expressing OLFML3-Myc/Flag. The cells are treated with 100 ng/mL LPS for 12 h. Cell lysate is immunoprecipitated with a sequential two-step affinity purification with Myc and Flag magnetic beads and then immunoblotted with an IRG1 antibody. (G) Validation of the interactions between IRG1-HA and endogenous OLFML3 in a RAW264.7 cell line stably expressing IRG1-HA. The cells are treated with 100 ng/mL LPS for 12 h or 24 h. Cell lysate is immunoprecipitated with an affinity purification with HA magnetic beads and then immunoblotted with an OLFML3 antibody.

Figure 4

Olfml3 knockout alters MMP and mitochondrial ROS production in macrophages. (A, B) Evaluation of the effects of OLFML3 on LPS-stimulated MMP changes in RAW264.7 (A) and BMDMs (B) using JC-1 fluorescence probe. (C, D) Analysis of the effects of OLFML3 on total cellular ROS in RAW264.7 (C) and BMDMs (D) using DCFH-DA fluorescence probe. (E, F) Analysis of the effects of OLFML3 on mtROS in RAW264.7 (E) and BMDMs (F) in response to LPS treatment using MitoSOX fluorescence probe. Cells are collected at 12 h post 100 ng/mL LPS stimulation. MFI, median fluorescent intensity. The data represent mean ± SEM of at least three biological replicates. ns, not significant.

Figure 5

OLFML3 affects macrophage functions by involving in IRG1-mediated metabolic processes. (A, B) Intracellular itaconate (A) and succinate (B) levels in wild-type and Olfml3^-/- RAW264.7 cells treated with LPS for 6 h. (C, D) Intracellular itaconate (C) and succinate (D) levels in wild-type and Olfml3^-/- BMDMs treated with LPS for 6 h. (E) Changes of ATP levels in LPS-treated wild-type and Olfml3^-/- RAW264.7 cells over a course of 24 h. (F, G) Olfml3 overexpression rescue of damaged migration ability in Olfml3^-/- RAW264.7 cells, as evaluated by transwell assay (F) and MCP-1 expression (G). For F, transmembraned cells are counted from three microscopic fields (with 40× magnification) in each membrane (three independent biological replicates per group) at 24 h after treatment with 1 μg/mL LPS. One representative microscopic field of each group is shown (magnification, 20×; Scale bar, 150 μm). For G, MCP-1 mRNA expression is determined by RT-qPCR at 4 h after treatment with 100 ng/mL LPS. (H) Irg1 overexpression rescue of damaged migration ability in Olfml3^-/- RAW264.7 cells, as evaluated by MCP-1 expression. The mRNA expression of MCP-1 is determined by RT-qPCR at 4 h after treatment with 100 ng/mL LPS. Data represent the mean ± SEM of at least three biological replicates. ns, not significant.

Figure 6

Examination of the formation of OLFML3-IRG1 complex and the subcellular localization of OLFML3 and IRG1 proteins. (A, B) Immunofluorescence analysis of the mitochondrial co-localization of IRG1-HA with OLFML3-Myc/Flag (A) or ΔSP-OLFML3-Myc/Flag (B) in HeLa cells. Mitochondria are stained with 200 nM Mitotracker Deep Red FM. (C) Fluorescence imaging analysis of the subcellular localization of OLFML3-OFP fusion protein in HeLa cells, treated with 1 μg/mL LPS. (D) Immunofluorescence analysis of the subcellular localization OLFML3-Myc/Flag in HeLa cells. Cells are stained with Flag antibody and Hoechst 33342. (E) Snapshot image showing 3D-SIM acquisitions of the fluorescence in LPS-stimulated HeLa cells stably expressing OLFML3-OFP (red) and IRG1-GFP (green). (F) Live-cell fluorescence time-lapse microscopy of cultured HeLa cells stably expressing OLFML3-OFP (red), IRG1-GFP (green) and Mitotracker (blue), monitored over a course of 12 h with 1 μg/mL LPS stimulation. PBS treatment is used as a control for basal changes of co-localization over time. (G) WB analysis of the cytoplasmic and nuclear contents of OLFML3- Myc/Flag in RAW264.7 treated with 100 ng/mL LPS for indicated durations. C, cytoplasmic. N, nuclear. Lamin A/C, nuclear marker. β-tubulin, cytoplasm marker. (H, I) WB analysis of the cytoplasmic and mitochondrial fractions of OLFML3 and IRG1 in RAW264.7 cell line stably expressing OLFML3-Myc/Flag (H) and wild-type RAW264.7 cells (I). Cells are treated with 100 ng/mL LPS or PBS for 12 h. The mitochondrial and cytosolic fractions of cell lysates are immunoblotted with IRG1 antibody and Flag antibody (H) or OLFML3 antibody (I) respectively. C, cytoplasmic. M, mitochondrial. Tom20, mitochondrial marker. Caspase 3, cytoplasm marker. (J) Evaluation of the sub-mitochondrial localization of OLFML3 in RAW264.7 cells stably expressing OLFML3-Myc/Flag. At 12 h after treatment with 100 ng/mL LPS, isolated mitochondrial fraction is treated with 100 ng/mL proteinase K in the absence or presence of 1% TritonX-100 for 20 min on ice and immunoblotted with antibodies as indicated.

Figure 7

OLFML3 facilitates IRG1 mitochondrial localization via AIFM1. (A, B) Examination of the effects of Olfml3 knockout on the level of mitochondrial IRG1 protein in RAW264.7 cells (A) and BMDMs (B). Cells are stimulated with 100 ng/mL LPS for 12 h and then fractionated. (C) Confocal immunofluorescence images showing the subcellular localization of IRG1-HA in wild type and Olfml3^-/- BMDMs following 100 ng/mL LPS stimulation for 12 h. The bar plot shows the quantification of co-localized IRG1 (green) and mitochondria (red). The fluorescence images in this figure represent one of the three independent replicates. Scale bars, 5 μm. (D) Identification of AIFM1 as an OLFML3 interacting protein in RAW264.7 by CoIP-MS. The top 5 candidate proteins are shown. (E, F) Analysis of the interactions between AIFM1 with OLFML3-Strep (E) or CC domain-Strep (F) in RAW264.7 stable cells line. The cells stably expressing OLFML3-Strep and CC domain-Strep are treated with PBS or 100 ng/mL LPS for 12 h. The cell lysate is immunoprecipitated with Strep tag II magnetic beads and then immunoblotted with antibodies as indicated. (G) Confirmation of the interaction between OLFML3, IRG1 and AIFM1 with endogenous co-IP assay. (H) Evaluation of the presence of AIFM1 in OLFML3-IRG1 complex by native PAGE in RAW264.7 cells stably expressing OLFML3-Strep. (I) Investigation of the presence of AIFM1 in OLFML3-IRG1 complex by 2D SDS-PAGE. (J) Confirmation of the expression of IRG1, OLFML3-Strep and AIMF1 in RAW264.7 cells stably expressing OLFML3-Strep. (K) Evaluation of the presence and the molecular weight of OLFML3-IRG1-AIFM1 complex using size exclusion chromatography. The anticipated molecular weight of OLFML3-Strep, IRG1 and AIFM1 are 51, 53 and 67 kD respectively. (L, M) Comparison of the effects of Olfml3^-/-Irg1^-/- and Olfml3^-/-Aifm1^-/- double knockout with Olfml3^-/- single knockout in LPS-treated RAW264.7 cells, as characterized by transwell assay (L) and MCP-1 mRNA level (M).

Figure 8

Schematic mechanisms of the modulatory function of OLFML3 in macrophages. OLFML3 facilitates IRG1mitochondrial localization via a mitochondrial transporter protein AIFM1 in macrophages. Depletion of OLFML3 in LPS-induced macrophages leads to decreased IRG1 mitochondrial localization and itaconate metabolic processes, and causes MMP loss and mtROS over-production, which in turn suppresses macrophage phagocytosis and migration and results in aggravated ALI.