Mmp12 Is Translationally Regulated in Macrophages during the Course of Inflammation

Abstract

Despite the importance of rapid adaptive responses in the course of inflammation and the notion that post-transcriptional regulation plays an important role herein, relevant translational alterations, especially during the resolution phase, remain largely elusive. In the present study, we analyzed translational changes in inflammatory bone marrow-derived macrophages upon resolution-promoting efferocytosis. Total RNA-sequencing confirmed that apoptotic cell phagocytosis induced a pro-resolution signature in LPS/IFNγ-stimulated macrophages (Mϕ). While inflammation-dependent transcriptional changes were relatively small between efferocytic and non-efferocytic Mϕ; considerable differences were observed at the level of de novo synthesized proteins. Interestingly, translationally regulated targets in response to inflammatory stimuli were mostly downregulated, with only minimal impact of efferocytosis. Amongst these targets, pro-resolving matrix metallopeptidase 12 (Mmp12) was identified as a translationally repressed candidate during early inflammation that recovered during the resolution phase. Functionally, reduced MMP12 production enhanced matrix-dependent migration of Mϕ. Conclusively, translational control of MMP12 emerged as an efficient strategy to alter the migratory properties of Mϕ throughout the inflammatory response, enabling Mϕ migration within the early inflammatory phase while restricting migration during the resolution phase.

Keywords: Mmp12; efferocytosis; inflammation; macrophage; resolution; translation.

Figure 1

Impact of efferocytosis on inflammatory responses in Mϕ. (A) Bone marrow-derived Mϕ (BMDMs) and NIH-3T3 caspase activatable (CA) cells were treated with 10 nM dimerizer for 6 and 24 h, stained with Annexin V and propidium iodide (PI), and analyzed using flow cytometry (n = 3). (B) BMDMs were stained with MitoTracker red, and apoptotic NIH-3T3 CA cells (after 6 h treatment with 10 nM dimerizer) were stained with pHrodo for 1 h prior to co-culture at a 1:2 or 1:5 ratio. Efferocytosis was followed by tracking double-positive cells in an Incucyte live cell analysis system for 24 h (n = 3). (C) BMDMs were stained with MitoTracker red, and apoptotic NIH-3T3 CA cells (after a 6 h treatment with 10 nM dimerizer) were stained with CFSE prior to co-culture at a 1:5 ratio for 2 or 16 h. Efferocytic Mϕ were assessed by flow cytometry (n = 3). (D,E) BMDMs were co-cultured with or without apoptotic NIH-3T3 CA cells (Eff) (after 6 h dimerizer treatment) at a 1:5 ratio for 16 h, prior to stimulation with 100 ng/mL LPS and 100 U/mL IFNγ for 6 h (D) or 24 h (E) (LPS). CD45⁺ Mϕ were purified by magnetic-activated cell sorting (MACS) and mRNA expression was quantified by RT-qPCR analysis. mRNA expression was normalized to Tbp and is given relative to untreated control Mϕ (n ≥ 5). Data are presented as means ± SEM and were statistically analyzed using two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to untreated control Mϕ; ^# p < 0.05, ^## p < 0.01 compared to LPS/IFNγ-treated control Mϕ.

Figure 2

Differential mRNA expression changes in response to inflammatory stimulation between efferocytic and non-efferocytic Mϕ. BMDMs were co-cultured with or without apoptotic NIH-3T3 CA cells (Eff) (after 6 h dimerizer treatment) at a 1:5 ratio for 16 h prior to stimulation with 100 ng/mL LPS and 100 U/mL IFNγ for 6 h (LPS). CD45⁺ Mϕ were purified by MACS-sorting followed by total RNA-seq analysis (n = 2). (A) Normalized read counts of differentially expressed genes (DEGs) (padj < 0.05) were visualized in a heatmap (z-score normalized counts) and categorized into clusters I, II, III, and IV by k-means clustering. Annotation columns depict the log2 fold change (L2FC) of DEGs. (B) Top five functional annotation clusters for each cluster as identified by DAVID [23,24]. (C) Gene set enrichment analysis (GSEA) of LPS/IFNγ-stimulated naïve vs. efferocytic Mϕ (p < 0.1, FDR < 0.1; NES = normalized enrichment score).

Figure 3

Differential de novo proteomic changes in response to inflammatory stimulation between efferocytic and non-efferocytic Mϕ. BMDMs were co-cultured with or without apoptotic NIH-3T3 CA cells (Eff) (after 6 h dimerizer treatment) at a 1:5 ratio for 16 h before stimulation with 100 ng/mL LPS and 100 U/mL IFNγ for 6 h (LPS). CD45⁺ Mϕ were purified by MACS-sorting followed by multiplexed enhanced protein dynamics proteomics (mePROD) (n = 3). (A) Normalized de novo peptide counts of differentially expressed peptides (DEPs) (padj < 0.05) were visualized in a heatmap (z-score normalized counts) and categorized into clusters I, II, III, and IV by k-means clustering. Annotation columns depict log2 fold change (L2FC) of DEPs. (B) Top five functional annotation clusters for each cluster as identified by DAVID [23,24].

Figure 4

Selection of translationally regulated targets in inflammatory Mϕ. (A) For the selection of candidates predominantly regulated at the translation level upon inflammatory stimulation (100 ng/mL LPS and 100 U/mL IFNγ, 6 h), substantially expressed targets (normalized read/peptide counts > 50) were filtered for pronounced LPS/IFNγ-selective regulation of de novo proteins (|FC_{Ctrl vs. LPS}| > 2; |FC_{Ctrl vs. Eff}| < 1.5) with minimal regulation at the mRNA level (|FC_{Ctrl vs. LPS}| < 1.2). Normalized read (left columns) and de novo peptide counts (right columns) of the top ten selected targets, sorted by Ctrl de novo peptide counts, are shown. (B) Abundance of MMP12 de novo peptides (upper panel) and total peptides (lower panel) based on de novo synthesis proteomics data. Data are presented as means ± SEM and were statistically analyzed using two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01 compared to untreated control Mϕ; ^## p < 0.01 compared to untreated efferocytic Mϕ.

Figure 5

Translational regulation of matrix metallopeptidase 12 (Mmp12) in inflammatory Mϕ. BMDMs were co-cultured with or without apoptotic NIH-3T3 CA cells (Eff) (after 6 h dimerizer treatment) at a 1:5 ratio for 16 h prior to stimulation with 100 ng/mL LPS and 100 U/mL IFNγ for 6 or 24 h (LPS). For further analyses, CD45⁺ Mϕ were purified by MACS-sorting. (A) Mmp12 mRNA expression was quantified by RT-qPCR analysis, normalized to Tbp, and presented relative to untreated control Mϕ (n ≥ 5). (B) MMP12 protein expression was analyzed by western blot analysis, normalized to total protein, and presented relative to untreated control Mϕ (n ≥ 5). (C,D) Translational status of Mmp12 was assessed by polysomal fractionation analysis. (C) UV profiles identified sub-polysomal (sub), early, and late polysomal fractions (representative tracks of three independent experiments are shown). (D) Gapdh (left panel) and Mmp12 mRNA (right panel) distribution across the gradients was analyzed by RT-qPCR (n = 3). (E) Secreted MMP12 protein was quantified in Mϕ supernatants by ELISA and is presented relative to untreated control Mϕ (n ≥ 5). (F) Net protein expression of MMP12 was calculated by combining mean intra- and extracellular MMP12 protein expression and is presented relative to untreated control Mϕ (n ≥ 5). Data are presented as means ± SEM and were statistically analyzed using two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to untreated control Mϕ; ^## p < 0.01 compared to LPS/IFNγ-treated control Mϕ.

Figure 6

Impact of MMP12 on Mϕ migration. BMDMs were transfected with Mmp12 or Ctrl siRNA (50 nM) for 24, 48, or 72 h. (A) Mmp12 mRNA expression was quantified by RT-qPCR analysis, normalized to Tbp, and presented relative to siCtrl-transfected Mϕ (72 h) (n = 3; upper panel). MMP12 protein expression was analyzed by Western blot analysis, normalized to total protein stain, and presented relative to siCtrl-transfected Mϕ (72 h) (n = 3; lower panel). (B,C) Mϕ were seeded 48 h after transfection on Matrigel (0.5×)/elastin (50 µg/mL) coated plates. (B) Mϕ were treated with recombinant MMP12 (r-MMP12, 50 ng/mL) 24 h after seeding, and migration was determined by live cell tracking for 24 h and quantified using the ImageJ manual tracking plugin. Representative tracks of siCtrl and siMmp12 in the presence or absence of r-MMP12 are depicted (upper panel). The migrated distance of 20 randomly selected cells per field of view was analyzed per replicate (n = 3; lower panel). (C) siCtrl-transfected Mϕ were stimulated with 100 ng/mL LPS and 100 U/mL IFNγ (LPS) 24 h after seeding, and migration was determined by live cell tracking for 24 h (n = 3). Data are presented as means ± SEM and were statistically analyzed using two-way ANOVA with Tukey’s multiple comparisons test (A,B) or unpaired t-test (C); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to untreated, siCtrl-transfected Mϕ. (D) Schematic model of the translational repression of MMP12 in Mϕ by pro-inflammatory LPS/IFNγ stimulation, resulting in reduced MMP12 protein levels and enhanced Mϕ migration in the local environment.